Human antibodies against eotaxin and their use

ABSTRACT

Specific binding members directed to eotaxin-1, in particular human antibodies and antibody fragments against human eotaxin-1 and especially those which neutralise eotaxin-1 activity. The antibodies VH and/or VL domain of the scFv fragment herein termed CAT-212 and of the IgG4 antibody herein termed CAT 213. One or more complementary determing regions (CDRs) of the CAT-212/-213 VH and/or VL domains, especially VH CRD3 in other antibody framework regions. Compositions containing specific binding members, and their use in methods of inhibiting or neutralising eotaxin, including methods of treatment of the human or animal body by therapy.

[0001] The present invention relates to specific binding membersdirected to eotaxin-1, in particular human antibodies against humaneotaxin-1 and especially those which neutralise eotaxin-1 activity.Preferred embodiments of the present invention employ the antibody VHand/or VL domain of the scFv fragment herein termed CAT-212 and of theIgG4 antibody herein termed CAT-213. Further preferred embodimentsemploy one or more complementarity determining regions (CDRs) of theCAT-212/-213 VH and/or VL domains, especially VH CDR3 in other antibodyframework regions. Further aspects of the present invention provide forcompositions containing specific binding members of the invention, andtheir use in methods of inhibiting or neutralising eotaxin, includingmethods of treatment of the human or animal body by therapy.

[0002] Eotaxin-1 is a chemoattractant protein that binds to a specificreceptor, CCR3, which is expressed predominantly on eosinophils. Ananti-eotaxin-1 antibody may be used to inhibit eosinophilia and therecruitment of eosinophils to sites of inflammation. In one embodiment,the present invention provides a human antibody fragment, named CAT-212,which was derived from a scFv phage display library. CAT-212 potentlyneutralises human eotaxin, with an IC₅₀ of 650 pM in a functionallyrelevant (chemotaxis) bio-assay. CAT-212 is of high affinity with aK_(D) of 15 pM. In a further embodiment, wherein the CAT-212 scFv isreformatted as a human IgG4, the antibody has been named CAT-213.CAT-213 is of similar potency to CAT-212 and neutralises human eotaxin,with an IC₅₀ of 700 pM in the chemotaxis assay. CAT-213 also blocksmononuclear cell chemotaxis in ovalbumin sensitised mice. Both CAT-212and CAT-213 potently block eosinophilia in an in vivo model of allergicinflammation.

[0003] Eosinophils normally account for 1B3% of the total peripheralblood leukocytes. A marked accumulation of eosinophils, a conditionknown as eosinophilia, can occur in many disorders such as allergicdiseases, parasitic infections and cancer (Rothenburg 1998).Eosinophilia is classified as having greater than 350 eosinophils percubic millimeter of blood, and in severe cases levels may rise to over5000 cells per cubic millimeter. As well as accumulating in theperipheral blood of the diseased individual, eosinophils may alsoselectively accumulate in any tissue in the body. Such eosinophilia canbe harmful due to the pro-inflammatory effects of the eosinophils. Ineosinophilic conditions such as asthma, there is frequently acorrelation between the number of infiltrating eosinophils and severityof the disease.

[0004] Eosinophils accumulate at inflammatory sites where they cansurvive for prolonged periods, depending on the combination of cytokinesproduced in their immediate environment. Eosinophils contain many toxicinflammatory mediators that are stored in granules. Upon activation byone or more of a wide number of cytokines, eosinophils degranulate torelease these toxins that include cationic proteins, such as major basicprotein, eosinophil-derived neurotoxin and eosinophilic peroxidase. Inaddition, activated eosinophils also release chemoattractants, lipidmediators such as leukotrienes and a wide range of inflammatorycytokines. Many of these substances have significant cytotoxic effectson tissues, such as the respiratory epithelium in asthma (Rothenberg,1998).

[0005] Chemokines are a group of homologous 8B10 kDa proteins (Luster,1998) that are subdivided into families based on the relative positionsof the conserved cysteine residues. Chemokines play an important role inmediating leukocyte extravasation from the blood into tissues as theyprovide the directional signals for the movement of leukocytes duringnormal development and homeostasis, and importantly, in inflammation.Although there are numerous chemotactic substances, such as leukotrieneB₄, interleukins and bacterial products, that are able to recruiteosinophils to tissues, only the chemokine, eotaxin-1 has been shown torecruit eosinophils specifically.

[0006] Human eotaxin is a member of the rapidly expanding group of β orCC (Cys-Cys) subfamily of chemokines. This group of molecules ischaracterised by the presence of 4 conserved cysteines, the first 2 ofwhich are adjacent and share a sequence identity between 20 and 75%.Members of this family include eotaxin-2 (Forssmann et al, 1997; Whiteet al, 1997), eotaxin-3 (Shinkai et al, 1999), monocyte chemoattractantprotein (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5 (Van Coillie et al, 1999),macrophage inflammatory protein (MIP)-1, MIP-1β, TARC, LARC, I309 andRANTES.

[0007] Eotaxin-1 is an 8.4 kDa, 74 amino acid protein that was firstdetected in the bronchoalveolar lavage (BAL) fluid from allergenchallenged sensitised guinea pigs (Griffiths-Johnson et al, 1993; Joseet al, 1994a). The molecule was first identified as a potentchemoattractant as it induced a substantial accumulation of eosinophilsat its intradermal injection site. The guinea pig gene was the firstcloned (Jose et al, 1994b, Rothenberg et al, 1995a), followed by mouse(Rothenberg et al, 1995b). The human eotaxin gene was subsequentlyidentified (Kitaura et al 1996; Garcia-Zepeda et al 1996; Ponath et al,1996) and the rat homologue has more recently been cloned (Williams etal, 1998). Human eotaxin has 61% identity with mouse and guinea pigeotaxin, and 62% identity with rat eotaxin. The human gene is located onchromosome 17 and comprises of three exons and two introns. The 5′flanking region of the gene contains a number of consensus regulatoryelements, including binding sites for AP-1, NFB, interferon gammaresponse element and the glucocorticoid receptor, suggesting that geneexpression is regulated by cytokines as well as by glucocorticosteroids.

[0008] Eotaxin can be produced by a variety of normal cell typesincluding epithelial cells, fibroblasts, endothelial cells,T-lymphocytes, monocytes and macrophages (Cook et al, 1998; Ponath etal, 1996a; Li et al, 1997). Although eosinophils are the main effectorcells for eotaxin, eosinophils also synthesize eotaxin themselves andstore it in intracellular granules (Nakajima et al, 1998). The releaseof eotaxin from eosinophils may contribute to the local accumulation ofeosinophils in inflammatory conditions. Eotaxin expression can beinduced from the-different cell types by many pro-inflammatorymediators, such as tumour necrosis factor-alpha, interferon andinterleukin-1.

[0009] Eotaxin-2 has recently been cloned (Forssmann et al, 1997; Whiteet al, 1997). It does not exhibit close sequence homology with eotaxin,as it shares only 39% amino acid identity. Like eotaxin, however,eotaxin-2 is a chemoattractant for eosinophils and basophils, albeit upto 10Bfold less potent. Eotaxin-3 has also been recently been identified(Shinkai et al, 1999) but its potency also appears to be 10Bfold lessthan that observed for eotaxin. Consequently, eotaxin-3 is chemotacticfor eosinophils and basophils only at relatively high concentrations(Kitaura et al, 1999).

[0010] In general, there is substantial redundancy in the binding ofchemokines to chemokine receptors. Typically, several different CCchemokines are able to bind a single chemokine receptor, and conversely,a single CC chemokine can bind to several different chemokine receptors.The chemokine receptor, CCR3, has many ligands including eotaxin, MCP-2,MCP-3, MCP-4, RANTES, eotaxin-2 and 3. Of these, eotaxin appears to bethe most important. Many of the ligands, such as MCP-2, MCP-3 andRANTES, have a relatively low affinity for CCR3 and are therefore notparticularly effective at inducing CCR3 mediated events. In contrast,eotaxin binds to the CC chemokine receptor 3 (CCR3) with relatively highaffinity, Kd=0.52 nM (Ponath et al, 1996a). Furthermore, eotaxin isunusual among CC chemokines in that it only binds to CCR3 and not to anyother chemokine receptor, that is, eotaxin is specific for CCR3.

[0011] Human CCR3 has been cloned (Combadiere et al, 1995; Daugherty etal, 1996) and is a 355 amino acid, 41 kDa, seven transmembrane domainprotein. It contains four cysteines in its extracellular domain andeight serine/threonine residues in the cytoplasmic tail that arepotential sites for G-protein mediated phosphorylation. CCR3 has nopotential sites for N-linked glycosylation. The human receptor bindsboth mouse and human eotaxin with equal affinity (Daugherty et al,1996). Mouse (Gao et al, 1996) and guinea pig (Sabroe et al, 1998) CCR3have subsequently been cloned and share 69 and 67% amino acid identitywith human CCR-3, respectively.

[0012] Human CCR-3 is principally expressed on eosinophils (Ponath etal, 1996b) and basophils (Uguccioni et al 1997; Yamada et al 1997). Itis also found on T_(H)2-type T cells (Sallusto et al, 1997), microglialcells in the central nervous system (He et al, 1997) and dendritic cells(Rubbert et al, 1998). Eotaxin is a chemoattractant and activator ofCCR3 expressing cells. On binding CCR3 on eosinophils, eotaxin causesintracellular calcium mobilisation, initiation of intracellular actinpolymerisation, upregulation of integrin expresssion and the inductionof oxygen radical production (Tenscher et al, 1996; Elsner et al, 1996).CCR3 is expressed at particularly high levels on eosinophils with 40,000(Daugherty et al, 1996) to 400,000 (Ponath et al, 1996b) receptors percell. Many CCR3 ligands, such as MCP-2, MCP-3, MCP-4 and RANTES, alsobind chemokine receptors other than CCR3 and can therefore mediatechemoattraction of a wide variety of cell types. In contrast, due to itshigh selectivity for CCR3, eotaxin is able to specifically chemoattractand activate CCR3 expressing cells such as eosinophils.

[0013] There is a growing body of evidence that blocking the effects ofeotaxin may used therapeutically. There are several in vivo studies thathave used either rabbit or rodent antibodies. One such study looked atthe effects of an intraveneously (iv) administered anti-eotaxinantibody. Gonzalo et al (1996) injected 20 μg an-anti-eotaxin rabbitpolyclonal antiserum iv into ovalbumin-challenged mice. Antibodyadministration prior to challenge reduced the eosinophilia by 56%, asmeasured by the number of eosinophils accumulating in broncho-alveolarlavage (BAL) fluid.

[0014] There are also a number of reports of the effects of locallyadministered anti-eotaxin antibodies. Humbles et al (1997) described theco-injection of guinea pig eotaxin (10 ng) with a rabbit polyclonalanti-eotaxin antiserum (10 μl) into the skin of naive guinea pigs thathad received a prior injection of ¹¹¹In-labelled eosinophils. Thepolyclonal antibody was able to completely block local eosinophilaccumulation. Similarly, Teixeira et al (1997) used a mouse model ofeosinophilia, in which murine eotaxin (1-30 pmol) was co-injected with arabbit polyclonal anti-eotaxin antiserum intradermally into the sites of4-8 hour active cutaneous anaphylactic reactions. Dilutions of 5% and20% of the antiserum blocked eosinophil recruitment by 45% and 95%,respectively. In addition, Sanz et al (1998) have looked at eosinophilaccumulation due to endogenously generated eotaxin induced byintradermal IL-4 injection. An anti-eotaxin polyclonal antiserum gave a54% inhibition of the late phase (24B28 hr) but not the early phase (0B4hr) of the response to IL-4.

[0015] To further understand the role of eotaxin in the healthy andeosinophil-mediated disease state, targeted gene disruption has beenused to generate mice that are deficient in eotaxin (Rothenberg et al1997). When these mice are sensitised and challenged with ovalbumin,eosinophil numbers were reduced by 70% in BAL from lungs of eotaxin nullmice compared with wild type mice (18 hrs after challenge). Thisdemonstrates that eotaxin enhances the magnitude of the eosinophilrecruitment after antigen challenge in models of asthma. Nakamura et al.(Am. J. Resp. & Crit. Care Med. (1999) 160: 1952-1956) demonstratesassociation of eotaxin levels with asthma and inverse relation with lungfunction.

[0016] Eotaxin mRNA is constitutively produced by a number of tissues,where it has been suggested to play a role in eosinophil homing(Rothenberg et al 1995). In the eotaxin null mice, no gross histologicalabnormalities could be detected in any organ, including those known toexpress eotaxin. Similarly no changes in leukocyte phenotype could bedetected. However, the total eosinophil count was reduced by 3-fold inthe null mice compared to the wild-type, suggesting that eotaxin alsoplays a role in determining the baseline number of eosinophils in theperipheral circulation (Rothenberg et al 1997).

[0017] Specific binding members according to the present invention areuseful-in binding to and preferably neutralising eotaxin, withtherapeutic potential in various diseases and disorders in which eotaxinplays a role. Exemplary diseases and disorders are discussed furtherbelow.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows neutralisation potency of scFv 3G3 in aneotaxin-mediated chemotaxis assay, described below. Data represent themean with standard error bars of two separate experiments. Maximalchemotaxis is the number of cells migrating through to the lower chamberin response to 50 ng/ml human eotaxin. The IC₅₀ for scFv 3G3 is 800 nM.

[0019]FIG. 2 shows CAT-212 specificity ELISA, with no signal abovebackground (PBS) on any of the other related or unrelated antigenstested. A weak signal can be observed against mouse eotaxin.

[0020]FIG. 3 shows neutralisation potency of CAT-212 and CAT-213 in aneotaxin-mediated chemotaxis assay.

[0021]FIG. 4 illustrates IC₅₀ of CAT-212 and CAT-213 in a competitionassay.

[0022]FIG. 5 shows a Scatchard plot of eotaxin binding to CAT-212, usedin determination of CAT-212 affinity for eotaxin.

[0023]FIG. 6 illustrates mouse eotaxin competition for binding toCAT-212.

[0024]FIG. 7 shows neutralisation by CAT-212 of the increase inintracellular Ca²⁺ concentration induced by eotaxin. Change influorescence measured over time in FLIPR in response to the addition of10 nM eoataxin±CAT-212 (concentration of CAT-212 shown in legend).Control is the addition of buffer alone. Addition of the Ab alone doesnot change the fluorescence significantly. The average of triplicatewells for eotaxin and duplicate wells for each antibody concentration isshown.

[0025]FIG. 8 shows area under the curve data for CAT-212 in a calciumflux assay, calculated for data from 12s to 100s. the lone point on they-axis is eotaxin alone. Average and std dev of triplicate wells foreotaxin and duplicated wells for each antibody concentration are shown.

[0026]FIG. 9 demonstrates specificity of binding of CAT-213 to humaneotaxin.

[0027]FIG. 10 shows the effect of CAT-212 and CAT-213 on human eotaxininduced eosinophil recruitment to the air pouch on ovalbumin sensitizedmice treated with IL-5. CAT-212 was administered i.po. whereas CAT-213was administered both i.po. and i.v. in separate experiments. The effectof antibody treatment was statistically evaluated by performing one wayANOVA with Dunnett's test using the differential cell count data.*P<0.05, **P<0.01 compared to human eotaxin challenged PBS controlanimals (=0% inhibition; n=7-8 mice). Each point represents the meanvalue and the vertical bars show SE. CAT-213 or CAT-212 administeredlocally to the air pouch caused a dose-related inhibition ofeosinophilia. CAT-213 given systemically also significantly inhibitedeosinophil chemotaxis.

[0028]FIG. 11 illustrates the effect of CAT-213 on ovalbumin inducedeosinophil recruitment to the air pouch on ovalbumin sensitized mice.CAT-213 was administered both i.po. and i.v. in separate experiments.The effect of antibody treatment was statistically evaluated byperforming one way ANOVA with Dunnett's test using the differential cellcount data. *P<0.05, **P<0.01 compared to ovalbumin challenged PBScontrol animals (=0% inhibition; n=7-8 mice). Each point represents themean value and the vertical bars show SE. CAT-213 administered locallyto the air pouch or given systemically caused a potent dose-relatedinhibition of eosinophilia. The effect of i.v. administration ofanti-mouse eotaxin IgG2A (R&D Systems mAb) on eosinophil recruitment areshown for comparison.

[0029]FIG. 12 illustrates the effect of CAT-213 on rhesus monkeyeotaxin- and murine eotaxin-induced chemotaxis of L1.2-CCR3 cells. Dataare expressed as mean±SEM from at least 3 experiments performed intriplicate or duplicate, respectively.

[0030]FIG. 13 shows neutralisation by CAT-213 of human eotaxin-inducedchemotaxis of human peripheral eosinophils. Data are expressed as mean±SEM from 3 experiments, performed with triplicate points.

[0031]FIG. 14 shows that CAT-213 inhibited eotaxin-mediated shape changeof human eosinophils. CAT-001 (the control antibody) was inactive. Dataare expressed as mean ±SEM from 5 experiments performed with duplicatepoints.

[0032] In one aspect, the present invention provides a specific bindingmember which binds human eotaxin and which comprises the CAT-212 VHdomain (SEQ ID NO. 2) and/or the CAT-212 VL domain (SEQ ID NO. 4)

[0033] Generally, a VH domain is paired with a VL domain to provide anantibody antigen binding site, although as discussed further below a VHdomain alone may be used to bind antigen. In one preferred embodiment,the CAT-212 VH domain (SEQ ID NO. 2) is paired with the CAT-212 VLdomain (SEQ ID NO. 4), so that an antibody antigen binding site isformed comprising both the CAT-212 VH and VL domains. In otherembodiments, the CAT-212 VH is paired with a VL domain other than theCAT-212 VL. Light-chain promiscuity is well established in the art.

[0034] One or more CDRs may be taken from the CAT-212 VH or VL domainand incorporated into a suitable framework. This is discussed furtherbelow. CAT-212 VH CDR's 1, 2 and 3 are shown in SEQ ID NO.'s 5, 6 and 7,respectively. CAT-212 VL CDR's 1, 2 and 3 are shown in SEQ ID NO.'s 8, 9and 10, respectively. Variants of the VH and VL domains and CDRs ofwhich the sequences are set out herein and which can be employed inspecific binding members for eotaxin can be obtained by means of methodsof sequence alteration or mutation and screening. Such methods are alsoprovided by the present invention.

[0035] Variable domain amino acid sequence variants of any of the VH andVL domains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), maybe less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than aboutalterations, 4, 3, 2 or 1. Alterations may be made in one or moreframework regions and/or one or more CDR's.

[0036] A specific binding member according to the invention may be onewhich competes for binding to antigen with any specific binding memberwhich both binds the antigen and comprises a specific binding member, VHand/or VL domain disclosed herein, or VH CDR3 disclosed herein, orvariant of any of these. Competition between binding members may beassayed easily in vitro, for example using ELISA and/or by tagging aspecific reporter molecule to one binding member which can be detectedin the presence of other untagged binding member(s), to enableidentification of specific binding members which bind the same epitopeor an overlapping epitope.

[0037] Thus, a further aspect of the present invention provides aspecific binding member comprising a human antibody antigen-binding sitewhich competes with CAT-212 or CAT-213 for binding to eotaxin.

[0038] Various methods are available in the art for obtaining antibodiesagainst eotaxin and which may compete with CAT-212 or CAT-213 forbinding to eotaxin.

[0039] In a further aspect, the present invention provides a method ofobtaining one or more specific binding members able to bind the antigen,the method including bringing into contact a library of specific bindingmembers according to the invention and said antigen, and selecting oneor more specific binding members of the library able to bind saidantigen.

[0040] The library may be displayed on the surface of bacteriophageparticles, each particle containing nucleic acid encoding the antibodyVH variable domain displayed on its surface, and optionally also adisplayed VL domain if present.

[0041] Following selection of specific binding members able to bind theantigen and displayed on bacteriophage particles, nucleic acid may betaken from a bacteriophage particle displaying a said selected specificbinding member. Such nucleic acid may be used in subsequent productionof a specific binding member or an antibody VH variable domain(optionally an antibody VL variable domain) by expression from nucleicacid with the sequence of nucleic acid taken from a bacteriophageparticle displaying a said selected specific binding member.

[0042] An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected specific binding membermay be provided in isolated form, as may a specific binding membercomprising such a VH domain. Ability to bind eotaxin may be furthertested, also ability to compete with CAT-212 or CAT-213 for binding toeotaxin. Ability to neutralise eotaxin may be tested, as discussedfurther below.

[0043] A specific binding member according to the present invention maybind eotaxin with the affinity of CAT-212 or CAT-213.

[0044] A specific binding member according to the present invention mayneutralise eotaxin with the potency of CAT-212 or CAT-213.

[0045] Binding affinity and neutralisation potency of different specificbinding members can be compared under appropriate conditions.

[0046] In addition to antibody sequences, a specific binding memberaccording to the present invention may comprise other amino acids, e.g.forming a peptide or polypeptide, such as a folded domain, or to impartto the molecule another functional characteristic in addition to abilityto bind antigen. Specific binding members of the invention may carry adetectable label, or may be conjugated to a toxin or enzyme (e.g. via apeptidyl bond or linker).

[0047] In further aspects, the invention provides an isolated nucleicacid which comprises a sequence encoding a specific binding member, VHor VL domains according to the present invention, and methods ofpreparing a specific binding member, a VH domain and/or a VL domain ofthe invention, which comprise expressing said nucleic acid underconditions to bring about production of said specific binding member, VHdomain and/or VL domain, and recovering it.

[0048] Specific binding members according to the invention may be usedin a method of treatment or diagnosis of the human or animal body, suchas a method of treatment (which may include prophylactic treatment) of adisease or disorder in a human patient which comprises administering tosaid patient an effective amount of a specific binding member of theinvention. Conditions treatable in accordance with the present inventioninclude those discussed elsewhere herein.

[0049] A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain and/or VLvariable domain disclosed herein.

[0050] Another aspect of the present invention provides nucleic acid,generally isolated, encoding a VH CDR or VL CDR sequence disclosedherein, especially a VH CDR selected from SEQ ID NO.'s 5, 6 and 7 or aVL CDR selected from SEQ ID NO.'s 8, 9 and 10, most preferably CAT-212VH CDR3 (SEQ ID NO. 7).

[0051] A further aspect provides a host cell transformed with nucleicacid of the invention.

[0052] A yet further aspect provides a method of production of anantibody VH variable domain, the method including causing expressionfrom encoding nucleic acid. Such a method may comprise culturing hostcells under conditions for production of said antibody VH variabledomain.

[0053] Analogous methods for production of VL variable domains andspecific binding members comprising a VH and/or VL domain are providedas further aspects of the present invention.

[0054] A method of production may comprise a step of isolation and/orpurification of the product.

[0055] A method of production may comprise formulating the product intoa composition including at least one additional component, such as apharmaceutically acceptable excipient.

[0056] These and other aspects of the invention are described in furtherdetail below.

[0057] Terminology

[0058] Specific Binding Member

[0059] This describes a member of a pair of molecules which have bindingspecificity for one another. The members of a specific binding pair maybe naturally derived or wholly or partially synthetically produced. Onemember of the pair of molecules has an area on its surface, or a cavity,which specifically binds to and is therefore complementary to aparticular spatial and polar organisation of the other member of thepair of molecules. Thus the members of the pair have the property ofbinding specifically to each other. Examples of types of specificbinding pairs are antigen-antibody, biotin-avidin, hormone-hormonereceptor, receptor-ligand, enzyme-substrate. This application isconcerned with antigen-antibody type reactions.

[0060] Antibody

[0061] This describes an immunoglobulin whether natural or partly orwholly synthetically produced. The term also covers any polypeptide orprotein having a binding domain which is, or is substantially homologousto, an antibody binding domain. Examples of antibodies are theimmunoglobulin isotypes and their isotypic subclasses; fragments whichcomprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; anddiabodies.

[0062] It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or othercell producing an antibody may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

[0063] As antibodies can be modified in a number of ways, the term“antibody” should be construed as covering any specific binding memberor substance having a binding domain with the required specificity.Thus, this term covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially synthetic. Chimeric molecules comprising an immunoglobulinbinding domain, or equivalent, fused to another polypeptide aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

[0064] It has been shown that fragments of a whole antibody can performthe function of binding antigens. Examples of binding fragments are (i)the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993). Fv, scFvor diabody molecules may be stabilised by the incorporation ofdisulphide bridges linking the VH and VL domains (Y. Reiter et al,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (S. Hu et al, Cancer Res., 56,3055-3061, 1996).

[0065] Diabodies are multimers of polypeptides, each polypeptidecomprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

[0066] Where bispecific antibodies are to be used, these may beconventional bispecific antibodies, which can be manufactured in avariety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol.4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas,or may be any of the bispecific antibody fragments mentioned above.Diabodies and scFv can be constructed without an Fc region, using onlyvariable domains, potentially reducing the effects of anti-idiotypicreaction.

[0067] Bispecific diabodies, as opposed to bispecific whole antibodies,may also be particularly useful because they can be readily constructedand expressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).

[0068] Antigen Binding Domain

[0069] This describes the part of an antibody which comprises the areawhich specifically binds to and is complementary to part or all of anantigen. Where an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by one or more antibody variabledomains (e.g. a so-called Fd antibody fragment consisting of a VHdomain). Preferably, an antigen binding domain comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH).

[0070] Specific

[0071] This may be used to refer to the situation in which one member ofa specific binding pair will not show any significant binding tomolecules other than its specific binding partner(s). The term is alsoapplicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

[0072] Comprise

[0073] This is generally used in the sense of include, that is to saypermitting the presence of one or more features or components.

[0074] Isolated

[0075] This refers to the state in which specific binding members of theinvention, or nucleic acid encoding such binding members, will be inaccordance with the present invention. Members and nucleic acid will befree or substantially free of material with which they are naturallyassociated such as other polypeptides or nucleic acids with which theyare found in their natural environment, or the environment in which theyare prepared (e.g. cell culture) when such preparation is byrecombinant. DNA technology practised in vitro or in vivo. Members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtitre platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.Specific binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NSO (ECACC85110503) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

[0076] By “substantially as set out” it is meant that the relevant CDRor VH or VL domain of the invention will be either identical or highlysimilar to the specified regions of which the sequence is set outherein. By “highly similar” it is contemplated that from 1 to 5,preferably from 1 to 4 such as 1 to 3 or 1 or 2, or 3 or 4,substitutions may be made in the CDR and/or VH or VL domain.

[0077] The structure for carrying a CDR of the invention will generallybe of an antibody heavy or light chain sequence or substantial portionthereof in which the CDR is located at a location corresponding to theCDR of naturally occurring VH and VL antibody variable domains encodedby rearranged immunoglobulin genes. The structures and locations ofimmunoglobulin variable domains may be determined by reference to(Kabat, E. A. et al, Sequences of Proteins of Immunological Interest.4th Edition. US Department of Health and Human Services. 1987, andupdates thereof, now available on the Internet(http://immuno.bme.nwu.edu)).

[0078] Preferably, a CDR amino acid sequence substantially as set outherein is carried as a CDR in a human variable domain or a substantialportion thereof. The VH CDR3 sequences substantially as set out hereinrepresent preferred embodiments of the present invention and it ispreferred that each of these is carried as a VH CDR3 in a human heavychain variable domain or a substantial portion thereof.

[0079] Variable domains employed in the invention may be obtained fromany germline or rearranged human variable domain, or may be a syntheticvariable domain based on consensus sequences of known human variabledomains. A CDR sequence of the invention (e.g. CDR3) may be introducedinto a repertoire of variable domains lacking a CDR (e.g. CDR3), usingrecombinant DNA technology.

[0080] For example, Marks et al (Bio/Technology, 1992, 10:779-783)describe methods of producing repertoires of antibody variable domainsin which consensus primers directed at or adjacent to the 5′ end of thevariable domain area are used in conjunction with consensus primers tothe third framework region of human VH genes to provide a repertoire ofVH variable domains lacking a CDR3. Marks et al further describe howthis repertoire may be combined with a CDR3 of a particular antibody.Using analogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of WO92/01047 so that suitable specific bindingmembers may be selected. A repertoire may consist of from anything from10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰members.

[0081] Analogous shuffling or combinatorial techniques are alsodisclosed by Stemmer (Nature, 1994, 370:389-391), who describes thetechnique in relation to a β-lactamase gene but observes that theapproach may be used for the generation of antibodies.

[0082] A further alternative is to generate novel VH or VL regionscarrying a CDR-derived sequences of the invention using randommutagenesis of one or more selected VH and/or VL genes to generatemutations within the entire variable domain. Such a technique isdescribed by Gram et al (1992, Proc. Natl. Acad. Sci., USA,89:3576-3580), who used error-prone PCR.

[0083] Another method which may be used is to direct mutagenesis to CDRregions of VH or VL genes. Such techniques are disclosed by Barbas etal, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al(1996, J. Mol. Biol. 263:551-567).

[0084] All the above described techniques are known as such in the artand in themselves do not form part of the present invention. The skilledperson will be able to use such techniques to provide specific bindingmembers of the invention using routine methodology in the art.

[0085] A further aspect of the invention provides a method for obtainingan antibody antigen binding domain specific for eotaxin antigen, themethod comprising providing by way of addition, deletion, substitutionor insertion of one or more amino acids in the amino acid sequence of aVH domain set out herein a VH domain which is an amino acid sequencevariant of the VH domain, optionally combining the VH domain thusprovided with one or more VL domains, and testing the VH domain or VH/VLcombination or combinations for to identify a specific binding member oran antibody antigen binding domain specific for a eotaxin antigen andoptionally with one or more of preferred properties, preferably abilityto neutralise eotaxin activity. Said VL domain may have an amino acidsequence which is substantially as set out herein.

[0086] An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

[0087] A further aspect of the invention provides a method of preparinga specific binding member specific for eotaxin antigen, which methodcomprises:

[0088] (a) providing a starting repertoire of nucleic acids encoding aVH domain which either include a CDR3 to be replaced or lack a CDR3encoding region;

[0089] (b) combining said repertoire with a donor nucleic acid encodingan amino acid sequence substantially as set out herein for a VH CDR3such that said donor nucleic acid is inserted into the CDR3 region inthe repertoire, so as to provide a product repertoire of nucleic acidsencoding a VH domain;

[0090] (c) expressing the nucleic acids of said product repertoire;

[0091] (d) selecting a specific binding member specific for a eotaxinantigen; and

[0092] (e) recovering said specific binding member or nucleic acidencoding it.

[0093] Again, an analogous method may be employed in which a VL CDR3 ofthe invention is combined with a repertoire of nucleic acids encoding aVL domain which either include a CDR3 to be replaced or lack a CDR3encoding region.

[0094] Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains which are then screened for a specificbinding member or specific binding members specific for eotaxin antigen.

[0095] A substantial portion of an immunoglobulin variable domain willcomprise at least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of NB or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailsbelow.

[0096] Although in a preferred aspect of the invention specific bindingmembers comprising a pair of VH and VL domains are preferred, singlebinding domains based on either VH or VL domain sequences form furtheraspects of the invention. It is known that single immunoglobulindomains, especially VH domains, are capable of binding target antigensin a specific manner.

[0097] In the case of either of the single chain specific bindingdomains, these domains may be used to screen for complementary domainscapable of forming a two-domain specific binding member able to bindeotaxin.

[0098] This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047 in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain specific binding memberis selected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Markset al, ibid.

[0099] Specific binding members of the present invention may furthercomprise antibody constant regions or parts thereof. For example, a VLdomain may be attached at its C-terminal end to antibody light chainconstant domains including human Cκ or Cλ chains, preferably Cλ chains.Similarly, a specific binding member based on a VH domain may beattached at its C-terminal end to all or part of an immunoglobulin heavychain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM andany of the isotype sub-classes, particularly IgG1 and IgG4. IgG4 ispreferred.

[0100] Specific binding members of the invention may be labelled with adetectable or functional label. Detectable labels include radiolabelssuch as ¹³¹I, or ⁹⁹Tc, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include enzyme labels such as horseradishperoxidase. Labels further include chemical moieties such as biotinwhich may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin.

[0101] Specific binding members of the present invention are designed tobe used in methods of diagnosis or treatment in human or animalsubjects, preferably human.

[0102] Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a specific binding member asprovided, pharmaceutical compositions comprising such a specific bindingmember, and use of such a specific binding member in the manufacture ofa medicament for administration, for example in a method of making amedicament or pharmaceutical composition comprising formulating thespecific binding member with a pharmaceutically acceptable excipient.

[0103] Clinical indications in which an anti-eotaxin antibody may beused to provide therapeutic benefit include asthma, eczema (atopicdermatitis) and other atopic diseases such as rhinitis, conjunctivitis,food allergy, allergic colitis which are recognised aseosinophil-mediated diseases. Experimental evidence favours eosinophilsas a cause of most cases of atopy so anti-eotaxin treatment is likely tobe effective for all these diseases. There are other allergicconditions, such as allergic bronchopulmonary aspergillosis and tropicaleosinophilia, that feature high peripheral eosinophil counts and whichmay be subject to anti-eotaxin treatment.

[0104] In particular, anti-eotaxin treatment in accordance with thepresent invention may be used to provide clear benefit for many patientswith asthma (Mattoli et al, 1997; Ying et al, 1997; Brown et al, 1998).About 10% of the population of the United Kingdom has asthma and currenttreatment is not entirely satisfactory: about 2000 deaths a year inEngland and Wales are attributed to asthma and about 6% of people withasthma are admitted to hospital (with asthmatic symptoms) each year.There is a clear need for improved treatment both for preventing asthmasymptoms and to treat more severe symptoms once they have developed.Anti-eotaxin treatment may be given orally, by injection (for example,subcutaneously or in emergencies, intravenously), by inhalation (tooptimise the profile of beneficial effects compared with any unwantedeffects) or by alternative routes of administration. The route ofadministration may be determined by the physicochemical characteristicsof the treatment, by special considerations for the disease, to optimiseefficacy or to minimise side-effects.

[0105] Skin conditions may best be treated with topical treatment withanti-eotaxin. Diseased skin often has increased absorptive capacity,compared with healthy skin, so topical treatment may well provide thebest route for therapy, where it is needed, without unwanted effectselsewhere in the body. If the skin condition covers much of the body, orif the disease is severe (maybe affecting other organs as well as theskin) then administration by injection or by other efficient means maybe more appropriate that the topical route. Local injection may beappropriate under certain circumstances (see the previous paragraph).

[0106] It is envisaged that anti-eotaxin treatment will not berestricted to use in the clinic. Patients may self-administer thetreatment and daily administration may be preferred over complex dosingschedules.

[0107] Combination treatments may be used to provide significantsynergistic effects, particularly the combination of an anti-eotaxinspecific binding member with one or more anti-interleukin-5 (IL-5)drugs. A specific binding member according to the present invention maybe provided in combination or addition to one or more corticosteroids,particularly one or more systemic corticosteroids. Combination treatmentwith one or more other anti-asthma/anti-allergy agents, especially otherApreventers@ such as cromoglycate, leukotriene (receptor) antagonists,xanthines and long-acting bronchodilators may be employed for asthmatreatment. Similar considerations of combinations apply to the use ofanti-eotaxin treatment for skin and other atopic conditions.

[0108] All forms of psoriasis, urticaria (including acute urticaria,chronic recurrent urticaria, delayed pressure urticaria, cold urticaria,dermographic urticaria), prurigo nodularis, papular erythematouseruptions, pemphigoid, porphyria cutanea tarda, persistent lightreaction, Wells' syndrome, eosinophilic cellulitis, drug eruptions,vasculitis (skin manifestation), purpura and other skin conditions maybe treated with anti-eotaxin in accordance with the present invention.These conditions can cover a large proportion of the body, may involveorgans other than the skin or may not cause the skin to have increasedpermeability. Even if effective applied topically, at the site ofaction, the preferred route may be systemic (through the body) for thesame considerations as suggested for atopic indications. Severe skindisease with associated systemic manifestations is a good example of asituation in which systemic treatment may be preferred to topicaltreatment or local injection.

[0109] Inflammatory bowel disease (ulcerative colitis and Crohn'sdisease) and eosinophilic colitis/enteritis/gastroenteritis/Shulman'ssyndrome may be treated effectively with an anti-eotaxin therapy.Eosinophils appear as a prominent cell-type in the lesions thatcharacterise these diseases.

[0110] Vasculitis of several forms, especially idiopathic, Hugues-Stovinsyndrome, Churg-Strauss syndrome, bronchocentric granulomatosis,eosinophilic pneumonitis (Löffler's syndrome), prolonged pulmonaryeosinophilia, Omenn's syndrome, Wiskott-Aldrich syndrome, familialeosinophilia and idiopathic hypereosinophilia may be treated withanti-eotaxin.

[0111] Eosinophilia of unknown cause can result complications such aspneumonitis, vasculitis, colitis, enteritis, gastroenteritis, Löffler'sendocarditis and heart valve fibrosis and many syndromes affectingconnective tissue. Eosinophilia can also be associated with malignantdisease (especially lymphomas, leukaemias and gastrointestinal cancers),drug treatments (eg cytokine infusions) and chronic fatigue syndrome.Anti-eotaxin treatment may be employed in any of these diseases.Similarly, eosinophilia-myalgia syndrome, toxic-oil syndrome, diffusefasciitis with eosinophilia (eosinophilic fasciitis) and eosinophilicmyositis may be treated with anti-eotaxin.

[0112] The eosinophil attraction caused by parasites may be a harmfuleffect so intervention with anti-eotaxin in these conditions may providebenefit. The diseases involving eosinophil attraction by pathogensinclude protozoal infection, and metazoan infections such as helmithinfestation and especially nematode infections (eg filariasis, hookworm,onchocerciasis, toxocariasis, ascariasis and trichinosis,angiostrongyliasis [eosinophilic meningitis]). Asymptomatic parasiticdisease may be the cause of many of the idiopathic forms ofeosinophil-mediated disease.

[0113] Anti-eotaxin treatment may have an effect on cells other thaneosinophils, e.g. those expressing CCR-3 such as basophils.

[0114] In accordance with the present invention, compositions providedmay be administered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, eg decisions on dosageetc, is within the responsibility of general practitioners and othermedical doctors. Appropriate doses of antibody are well known in theart; see Ledermann J. A. et al. (1991) Int J. Cancer 47: 659-664;Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates andRadiopharmaceuticals 4: 915-922.

[0115] The precise dose will depend upon a number of factors, includingwhether the antibody is for diagnosis or for treatment, the size andlocation of the area to be treated, the precise nature of the antibody(e.g. whole antibody, fragment or diabody), and the nature of anydetectable label or other molecule attached to the antibody. A typicalantibody dose will be in the range 0.5 mg to 100 g for systemicapplications, and 10 μg to 1 mg for local applications. Typically, theantibody will be a whole antibody, preferably the IgG4 isotype. This isa dose for a single treatment of an adult patient, which may beproportionally adjusted for children and infants, and also adjusted forother antibody formats in proportion to molecular weight. Treatments maybe repeated at daily, twice-weekly, weekly or monthly intervals, at thediscretion of the physician.

[0116] Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

[0117] Thus pharmaceutical compositions according to the presentinvention, and for use in accordance with the present. invention, maycomprise, in addition to active ingredient, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material willdepend on the route of administration, which may be oral, or byinjection, e.g. intravenous.

[0118] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may comprise a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally comprise a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0119] For intravenous injection, or injection at the site ofaffliction, the active ingredient will be in the form of a parenterallyacceptable aqueous solution which is pyrogen-free and has suitable pH,isotonicity and stability. Those of relevant skill in the art are wellable to prepare suitable solutions using, for example, isotonic vehiclessuch as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

[0120] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated. Other treatments may include theadministration of suitable doses of pain relief drugs such asnon-steroidal anti-inflammatory drugs (e.g. asprin, paracetamol,ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.

[0121] The present invention provides a method comprising causing orallowing binding of a specific binding member as provided herein toeotaxin. As noted, such binding may take place in vivo, e.g. followingadministration of a specific binding member, or nucleic acid encoding aspecific binding member, or it may take place in vitro, for example inELISA, Western blotting, immunocytochemistry, immuno-precipitation oraffinity chromatography.

[0122] The amount of binding of specific binding member to eotaxin maybe determined. Quantitation may be related to the amount of the antigenin a test sample, which may be of diagnostic interest, which may be ofdiagnostic interest.

[0123] The reactivities of antibodies on a sample may be determined byany appropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody. Bound antigen is physicallyseparated from unbound antigen and the amount of radioactive antigenbound to the antibody determined. The more antigen there is in the testsample the less radioactive antigen will bind to the antibody. Acompetitive binding assay may also be used with non-radioactive antigen,using antigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

[0124] Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

[0125] The signals generated by individual antibody-reporter conjugatesmay be used to derive quantifiable absolute or relative data of therelevant antibody binding in samples (normal and test).

[0126] The present invention also provides the use of a specific bindingmember as above for measuring antigen levels in a competition assay,that is to say a method of measuring the level of antigen in a sample byemploying a specific binding member as provided by the present inventionin a competition assay. This may be where the physical separation ofbound from unbound antigen is not required. Linking a reporter moleculeto the specific binding member so that a physical or optical changeoccurs on binding is one possibility. The reporter molecule may directlyor indirectly generate detectable, and preferably measurable, signals.The linkage of reporter molecules may be directly or indirectly,covalently, e.g. via a peptide bond or non-covalently. Linkage via apeptide bond may be as a result of recombinant expression of a genefusion encoding antibody and reporter molecule.

[0127] The present invention also provides for measuring levels ofantigen directly, by employing a specific binding member according tothe invention for example in a biosensor system.

[0128] The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

[0129] The present invention further extends to a specific bindingmember which competes for binding to eotaxin with any specific bindingmember which both binds the antigen and comprises a V domain including aCDR with amino acid substantially as set out herein or a V domain withamino acid sequence substantially as set out herein. Competition betweenbinding members may be assayed easily in vitro, for example by tagging aspecific reporter molecule to one binding member which can be detectedin the presence of other untagged binding member(s), to enableidentification of specific binding members which bind the same epitopeor an overlapping epitope. Competition may be determined for exampleusing the ELISA as described in Example 1.

[0130] In testing for competition a peptide fragment of the antigen maybe employed, especially a peptide including an epitope of interest. Apeptide having the epitope sequence plus one or more amino acids ateither end may be used. Such a peptide may be said to “consistessentially” of the specified sequence. Specific binding membersaccording to the present invention may be such that their binding forantigen is inhibited by a peptide with or including the sequence given.In testing for this, a peptide with either sequence plus one or moreamino acids may be used.

[0131] Specific binding members which bind a specific peptide may beisolated for example from a phage display library by panning with thepeptide(s).

[0132] The present invention further provides an isolated nucleic acidencoding a specific binding member of the present invention. Nucleicacid includes DNA and RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a CDR or VH or VL domain of theinvention as defined above.

[0133] The present invention also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes which compriseat least one polynucleotide as above.

[0134] The present invention also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyCDR, VH or VL domain, or specific binding member as provided itselfforms an aspect of the present invention, as does a method of productionof the encoded product, which method comprises expression from encodingnucleic acid therefor. Expression may conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression a VH or VL domain,or specific binding member may be isolated and/or purified using anysuitable technique, then used as appropriate.

[0135] Specific binding members, VH and/or VL domains, and encodingnucleic acid molecules and vectors according to the present inventionmay be provided isolated and/or purified, e.g. from their naturalenvironment, in substantially pure or homogeneous form, or, in the caseof nucleic acid, free or substantially free of nucleic acid or genesorigin other than the sequence encoding a polypeptide with the requiredfunction. Nucleic acid according to the present invention may compriseDNA or RNA and may be wholly or partially synthetic. Reference to anucleotide sequence as set out herein encompasses a DNA molecule withthe specified sequence, and encompasses a RNA molecule with thespecified sequence in which U is substituted for T, unless contextrequires otherwise.

[0136] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

[0137] The expression of antibodies and antibody fragments inprokaryotic cells such as E. coli is well established in the art. For areview, see for example Plückthun, A. Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a specific bindingmember, see for recent reviews, for example Ref, M. E. (1993) Curr.Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. OpinionBiotech 6: 553-560.

[0138] Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator sequences, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may beplasmids, viral e.g. ‘phage, or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

[0139] Thus, a further aspect of the present invention provides a hostcell containing nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

[0140] The introduction may be followed by causing or allowingexpression from the nucleic acid, e.g. by culturing host cells underconditions for expression of the gene.

[0141] In one embodiment, the nucleic acid of the invention isintegrated into the genome (e.g. chromosome) of the host cell.Integration may be promoted by inclusion of sequences which promoterecombination with the genome, in accordance-with standard techniques.

[0142] The present invention also provides a method which comprisesusing a construct as stated above in an expression system in order toexpress a specific binding member or polypeptide as above.

[0143] Aspects and embodiments of the present invention will now beillustrated by way of example with reference to the followingexperimentation.

[0144] Abbreviations

[0145] TES: 0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose;

[0146] 2TYAG: 2TY supplemented with 100 μg/ml ampicillin and 2% glucose;

[0147] 2TYAK: 2TY supplemented with 100 μg/ml ampicillin and 50 μg/mlkanamycin;

[0148] TMB: 3,3′,5,5′-Tetramethyl Benzidine;

[0149] ACE: 3-amino-9-ethyl-carbazole;

[0150] IC₅₀: 50% inhibitory concentration;

[0151] 6MPBS: 6×PBS containing 18% Marvel blocking solution;

[0152] A: Absorbance;

[0153] BSA: Bovine serum albumin;

[0154] BAL: Bronchoalveolar lavage;

[0155] CCR: CC Chemokine receptor;

[0156] CC: Cys-Cys;

[0157] DMEM: Dulbecco's Modified Eagles medium;

[0158] ELISA: Enzyme linked immunosorbent assay;

[0159] Fluo-3 AM: Fluo-3 acetoxymethyl aster;

[0160] FCS: Foetal calf serum;

[0161] gs: Glutamine synthetase;

[0162] V_(H): Heavy chain variable;

[0163] HRP: Horseradish peroxidase;

[0164] IMAC: Immobilised Metal Affinity Chromatography;

[0165] ICC: Immunocytochemistry;

[0166] Ig: Immunoglobulin;

[0167] IPTG: Isopropyl β-D-thiogalactopyranoside;

[0168] V_(L): Light chain variable;

[0169] MCP: Monocyte Chemoattractant Protein;

[0170] MOI: Multiplicity of infection;

[0171] Hepes: N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid];

[0172] nM: Nanomolar;

[0173] NHS: N-Hydroxysuccinimide;

[0174] NSO: Non-secreting mouse myeloma 0;

[0175] OCT: Optimum cutting tissue compound;

[0176] MPBS: PBS containing 3% Marvel;

[0177] PBS: Phosphate Buffered Saline;

[0178] PBST/0.05: Phosphate Buffered Saline+0.05% (v/v) Tween 20;

[0179] PBST: Phosphate Buffered Saline+0.1% (v/v) Tween 20;

[0180] pM: Picomolar;

[0181] PAGE: Polyacrylamide Gel Electrophoresis;

[0182] PCR: Polymerase Chain Reaction;

[0183] scFv: Single chain fragment variable;

[0184] SDS: Sodium dodecyl sulphate;

[0185] SELDI: Surface-enhanced laser desorption/ionization;

[0186] TNF-α: Tumour Necrosis Factor-alpha.

LIST OF EXPERIMENTAL EXAMPLES

[0187] EXAMPLE 1: Isolation of anti-human eotaxin scFvs

[0188] EXAMPLE 2: Neutralisation potency of scFv 3G3 in a chemotaxisassay

[0189] EXAMPLE 3: Derivation and sequence of CAT-212

[0190] EXAMPLE 4: Specificity of CAT-212

[0191] EXAMPLE 5: Neutralisation potency of CAT-212 in a chemotaxisassay

[0192] EXAMPLE 6: CAT-212 competition assay for eotaxin binding toCAT-212

[0193] EXAMPLE 7: Determination of CAT-212 affinity for eotaxin

[0194] EXAMPLE 8: Mouse eotaxin competition for binding to CAT-212

[0195] EXAMPLE 9: Neutralisation potency of CAT-212 in a calcium fluxassay

[0196] EXAMPLE 10: Immunoreactivity of CAT-212 with human nasal polyp

[0197] EXAMPLE 11: Conversion of CAT-212 to IgG4 format (CAT-213)

[0198] EXAMPLE 12: Neutralisation potency of CAT-213 in a chemotaxisassay

[0199] EXAMPLE 13: CAT-213 competition assay for eotaxin binding toCAT-212

[0200] EXAMPLE 14: Effects of CAT-212 and CAT-213 in an in vivo model ofallergic inflammation

[0201] EXAMPLE 15: Neutralisation potency of CAT-213 in aneotaxin-mediated chemotaxis assay using L1.2 CCR-3 transfected cells:rhesus monkey and mouse eotaxin

[0202] EXAMPLE 16: Neutralisation potency of CAT-213 in aneotaxin-mediated chemotaxis assay using human eosinophils

[0203] EXAMPLE 17: Neutralisation potency of CAT-213 in aneotaxin-mediated eosinophil shape change assay

EXAMPLE 1

[0204] Isolation of Anti-Human Eotaxin scFvs

[0205] scFv Antibody Repertoire

[0206] A large single chain Fv human antibody library (Vaughan et al,1996) derived from B-lymphocytes isolated from tonsil, bone marrow andperipheral blood and cloned into a phagemid vector was used forselections. This scFv repertoire is calculated to have ca. 1.3×10¹⁰individual recombinants. Antibodies from this repertoire alsoincorporate a C-terminal stretch of 6 histidines to enable scFvpurification by immobilised metal affinity chromatography (IMAC), and ashort sequence derived from c-myc to provide a generic detection systemusing the monoclonal anti-c-myc antibody, 9E10.

[0207] Selection of scFv

[0208] Human eotaxin (Cambridge BioSciences)) was coated at 10 μg/mleither directly onto immunotubes (Nunc; Maxisorp), or coupled toDisuccinimidyl suberate activated BSA coated onto Maxisorb microtitreplates (Nunc). For the first round of selection, 10¹² titered units oflibrary phage were used. Three rounds of selection of the scFv librarywere performed following standard panning protocols (Vaughan et al,1996). Individual clones from rounds 2 and 3 of selection were rescuedand screened by phage ELISA.

[0209] Rescue of Phage for ELISA

[0210] Individual colonies from rounds 2 and 3 of selection wereinoculated into 96-well plates containing 100 μl 2TY medium supplementedwith 100 μg/ml ampicillin and 2% glucose (2TYAG) per well. Plates wereincubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was addedto each well to an MOI of 10 and the plates were incubated. for afurther 1 hour at 37° C. The plates were centrifuged in a benchtopcentrifuge at 2000 rpm for 10 minutes. The supernatant was removed andcell pellets were resuspended in 100 μl 2TY supplemented with 100 μg/mlampicillin and 50 μg/ml kanamycin (2TYAK) and incubated at 30° C.overnight, shaking. Plates were centrifuged at 2000 rpm for 10 min andthe 100 μl phage-containing supernatant from each well recovered into a96-well plate. To block the phage, 20 μl of 6×PBS containing 18% Marvelblocking solution (6MPBS) was added to each well and incubated at roomtemperature for 1 hour. The phage are now ready to use in ELISA.

[0211] Phage ELISA

[0212] Flexible 96-well plates (Falcon) were coated overnight at 40° C.with 0.5 μg/ml human eotaxin in PBS, or with PBS alone as a control.After coating, the solutions were removed from the wells, and the plateswere blocked for 1 hour at room temperature in PBS containing 3% Marvel(MPBS). The plates were washed 3 times with PBS and then 50 μl ofpre-blocked phage was added to each well. The plates were incubatedstationary at room temperature for 1 hour. The plates were washed with 3changes of PBS containing 0.1% (v/v) Tween 20 (PBST) followed by 3changes of PBS at room temperature.

[0213] To each well, 50 μl of an anti-gene VIII-HRP conjugate(Pharmacia) at a 1 in 5000 dilution in MPBS was added and the platesincubated at room temperature for 1 hour. Each plate was washed 3×withPBST followed by 3×with PBS. Fifty μl of 3,3′,5,5′-Tetramethyl Benzidine(TMB; Sigma) substrate was then added to each well, and incubated atroom temperature for 30 minutes or until colour development. Thereaction was stopped by the addition of 25 μl of 0.5 M H₂SO₄. The signalgenerated was measured by reading the absorbance at 450 nm (A₄₅₀) usinga microtitre plate reader (Bio-Rad 3550).

[0214] Anti-Eotaxin scFvs

[0215] It was found to be unusually difficult to isolateeotaxin-specific scFvs, with only 4 different scFv being identified byphage ELISA. When the same scFv library has been selected against otherantigens, many more scFvs are typically identified. The clone identifiedfor further characterisation was named 3G3, and this clone consistentlygave signals on human eotaxin of 5-10 fold over that seen on PBS inELISA.

EXAMPLE 2

[0216] Neutralisation Potency of scFv 3G3 in an Eotaxin-MediatedChemotaxis Assay

[0217] Background

[0218] The neutralisation potency of the antiBeotaxin scFv 3G3 wasdetermined using an in vitro chemotaxis assay.

[0219] The chemotaxis assay is a particularly relevant in vitro potencyassay as it is the ability of eotaxin to chemoattract CCR3 expressingcells that it is desirable to inhibit in vivo. The assay works on theprinciple that cells expressing the CCR3 receptor will migrate towardsan eotaxin gradient by chemotaxis. The method detailed here is based onthat described by Ponath et al, 1996a. Briefly, eotaxin was placed inthe bottom well of a Transwell plate (Costar), along with the testantibody in an appropriate buffer. Transfected cells expressing the CCR3receptor were placed in the top chamber of the Transwell. The twochambers were separated by a polyester membrane with a pore size of 3μM. Cells only moved through the pores in response to a CCR3 ligand,such as eotaxin. After a defined incubation period, the number of cellsthat had migrated through to the bottom chamber were counted, and thisnumber is a measure of the chemotactic activity of the chemokine thatwas placed in the top chamber. Inhibition of this chemotactic activitycan therefore be assessed in this assay.

[0220] Maintenance of CCR3 Cells

[0221] L1.2 cells were transfected with the human CCR3 receptor togenerate a stable cell line expressing CCR3 on its surface. The cellswere maintained in RPMI-1640 (Sigma) containing 10% heat-inactivated FCS(Biowhittaket), 2% L-glutamine (Sigma), 10 U penicillin (Sigma), 100g/ml streptomycin (Sigma), 250 μg/ml kanamycin (Sigma) and 400 μg/mlGeneticin (Gibco). The cells were kept between 1B2×10⁶/ml for use in theassay. No stimulation of the cells to up-regulate CCR3 expression wasrequired for the chemotaxis assay. The response to 50 ng/ml eotaxin wascarefully monitored, as the number of migrating cells can decline withcell passage number, presumably due to alterations in the CCR3expression level. Typically, a 10% sample of the cells migrating throughto the lower chamber were subsequently quantitated by flow cytometry. Itwas found that 50 ng/ml eotaxin typically induced chemotaxis of8,000-10,000 cells (this is the 10% value; i.e. 80,000B100,000 cellsmigrated through to the lower chamber in total).

[0222] Chemotaxis Assay

[0223] The chemotaxis assay buffer comprises RPMI-1640 (Sigma)containing 1% endotoxin free BSA (Bayer Pentex), 100 U/ml penicillin,100 μg/ml streptomycin. Test solutions of antibody (in duplicate) werediluted to the desired concentration in assay buffer. A typical dilutionrange for IMAC-purified (see next section) scFv 3G3 was 100 μg/ml to 1μg/ml. Human eotaxin (AlbaChem) was added to a final concentration of 50ng/ml when mixed with the appropriate test scFv. All samples wereincubated for 30 minutes at room temperature. CCR3 L1.2 cells werecentrifuged at 1,200 rpm in an Heraeus Sepatech 1.0 benchtop centrifugefor 5 mins, the media removed by aspiration and the cells resuspended in25 ml of PBS. The cells were then re-centrifuged and the cell pelletresuspended in assay buffer to 10⁷ cells/ml. Test solutions (0.6 ml perwell) were placed into the bottom chambers of the Transwell plates.Transwells were placed over the test solutions and 100 μl of cells (10⁶cells total) placed into the top chamber of each transwell. Incubationwas for 4 hours at 37° C. under 5% CO₂. The plates were tapped gentlyprior to removal of the transwells, to dislodge any cells attached tothe underside of the membrane. Cells migrating through to the bottomchamber were resuspended and counted using a flow cytometer. Sampleswere each counted for 60 seconds using a medium flow rate. Thepercentage inhibition of chemotaxis caused by the test antibody was thendetermined.

[0224] Purification of scFv

[0225] To determine the potency of 3G3 scFv in the chemotaxis assay,scFv was first prepared by IMAC. 2TYAG (5 ml) was inoculated with asingle colony of 3G3 and grown overnight at 30° C., shaking. Thisovernight culture was then used to inoculate 500 ml of 2TY containing100 μg/ml ampicillin and 0.1% glucose, and grown at 30° C., shaking,until an A₆₀₀ of 1.0 was attained. Isopropyl β-D-thiogalactopyranoside(IPTG) was added to 1 mM and the culture was grown for a further 3.5hours at 30° C.

[0226] Cells were harvested by centrifugation at 5,000 rpm, andresuspended in 10 ml of TES (0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose;ice cold). A further 15 ml of a 1:5 dilution (in water) of TES wasadded, and the cell suspension incubated on a turning wheel at 40° C.for 30 minutes. This caused osmotic shock and yielded a periplasmicextract containing the scFv. Residual cells and debris were pelleted bycentrifugation at 9,000 rpm for 20 minutes at 40° C. The supernatant wastransferred to a new tube, and 50 μl of 1 M MgCl₂ added. Two ml of aNi—NTA slurry (Qiagen), pre-washed with buffer (50 mM sodium phosphate,pH 8, 300 mM NaCl) together with a protease inhibitor tablet (BoehringerMannheim) were then added to the periplasmic extract. The preparationwas incubated, rotating, overnight at 40° C. The Ni—NTA was pelleted bycentrifugation at 2,000 rpm for 5 minutes, and the supernatent wasaspirated. The agarose beads were washed 3 times with 50 ml wash buffer,centrifuging to collect the agarose in between each wash. Ten ml of washbuffer was added after the final wash, and the slurry was loaded on to apolyprep column (BioRad). Two ml elution buffer (50 mM NaPi, pH 8, 300mM NaCl, 250 mM imidazole) was added to the drained agarose, and theelutate was collected. IMAC purified scFv was buffer exchanged in to PBSby use of a Nap 5 column (Pharmacia) according to the manufacturer'sinstructions. The A₂₈₀ was read and the protein concentration determinedusing a molar extinction coefficient of 1 mg/ml protein=A₂₈₀ 1.4.Purified scFv was stored in 500 μl aliquots at −70° C.

[0227] Results

[0228] Typical data for purified scFv 3G3 in the chemotaxis assay isshown in FIG. 1. The IC₅₀ for scFv 3G3 is 800 nM. This antibody istherefore of low-moderate potency.

EXAMPLE 3

[0229] Derivation and Sequence of CAT-212

[0230] The low-moderate potency of scFv 3G3 makes this antibody arelatively unsuitable candidate for any therapeutic application.

[0231] A further scFv antibody, named CAT-212, was obtained using avariety of techniques and its DNA sequence determined.

[0232] DATA Sequencing

[0233] DNA was amplified by polymerase chain reaction (PCR) fromindividual colonies on 2TYAG agar plates using the vector-specificprimers pUC19reverse and fdtetseq (Vaughan et al, 1996). Amplificationconditions comprise 94° C. for 1 minute, 55° C. for 1 minute and 72° C.for 2 minutes, for 30 cycles, prior to a final 10 minute extension at72° C. The PCR products were purified using a PCR Clean-up Kit (Promega)in to a final volume of 50 [2l H₂O. Between 2 and 5 μl of each insertpreparation was used as the template for sequencing using the TaqDye-terminator cycle sequencing system (Applied Biosystems). The primers(Osbourn et al, 1996) gene3leader, PCRHLink were used to sequence theV_(H) and PCRLLink and mycseq10 to sequence the V_(L) of the scFv.

[0234] The nucleotide sequences of CAT-212 V_(H) and V_(L) are shown inSEQ ID NO.'s 1 and 3, respectively. The derived amino acid sequence ofCAT-212 V_(H) and V_(L) are shown in SEQ ID NO.'s 2 and 4, respectively.

[0235] The individual V_(H) and V_(L) segments of the antibodies werealigned to the known human germline sequences in V-BASE (Tomlinson etal, 1995) and the closest germline identified. The closest germline forthe heavy chain of CAT-212 was identified as DP49, a member of theV_(H)3 family. The CAT-212 V_(H) has just 6 changes from the DP49germline, two of these within CDR2. The closest germline for the lightchain of CAT-212 was identified as DP_(k)5, a member of the V_(k)1family. The CAT-212 V_(L) has only 2 changes from the DP_(k)5 germline,both within CDRs. The entire sequence of CAT-212, or any derivativethereof (such as CAT-213), has a total of only 8 changes from germline,4 of which are in CDRs. This should further minimise any possible riskof immunogenicity when these human antibodies are used to treatpatients.

EXAMPLE 4

[0236] Specificity of CAT-212

[0237] Two techniques were used to investigate the specificity ofCAT-212: phage ELISA and Western blotting.

[0238] Phage ELISA

[0239] To determine the specificity of CAT-212, a phage ELISA wasperformed against human and mouse eotaxin, and a panel of related andun-related human antigens; MIP-1α, MCP-1, MCP-2, MCP-3, MCP-4, IL-1α,IL-1β, IL-5, IL-18, IL-12, RANTES, transforming growth factor (TGF)-β1,TGF-β2, TNFα and PBS.

[0240] Individual E. coli colonies containing CAT-212 phagemid wereinoculated into 5 ml 2YTAG and incubated at 37□ C. for 4 hours, shaking.M13KO7 helper phage (Pharmacia) was added to each tube to an MOI of 10and incubated for 30 min at 37° C. for 1 hour, the first 30 minutesstatic and the final 30 minutes with gentle shaking. Cells were pelletedby centrifugation at 3,500 rpm for 10 minutes and the supernatantdiscarded. Cell pellets were resuspended in 5 ml 2TYAK and incubated at30° C. overnight with-shaking. The next day, the cells were pelleted bycentrifugation at 3,500 rpm for 10 minutes. The phage-containingsupernatant (5 ml) was carefully transferred to a fresh tube, 1 ml of6MPBS added, and incubated at room temperature for 1 hour to pre-blockthe phage prior to ELISA.

[0241] The coating concentrations and suppliers of the various antigensare shown in Table 1. ELISAs were performed essentially as described inExample 1. The results are shown in FIG. 9. CAT-212 is specific forhuman eotaxin and does not cross-react with any unrelated or relatedhuman antigen tested. A very slight signal over background was seen onmouse eotaxin. This may indicate that CAT-212 recognises mouse eotaxin,albeit relatively weakly compared to human. No commercially availableeotaxin could be obtained for any species other than human or mouse.

[0242] Western Blotting

[0243] Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) was performed using a PHAST system and 10B15% gels(Pharmacia). Samples of eotaxin and human MCP-1 (400 ng) were run on thegel, alongside molecular weight markers. Electrophoresis was performedthen proteins were transferred to an immobilon-P (Millipore) membrane bydiffusion. The membrane was blocked by incubation MPBS for 1 hour, andthen probed with 10 μg/ml IMAC-purified CAT-212 scFv or an irrelevantscFv in MPBS for 1 hour, shaking at room temperature. Following this,the membrane was washed (3×2 minutes) in PBST, and then incubated withbiotinylated anti-myc tag antibody (9E10) at 1 μg/ml for 1 hour. Afteranother wash, the blot was incubated with streptavidin-HRP (Pierce) at 1μg/ml for 1 hour, before a final wash. The blot was placed into ECLsubstrate (Amersham) and exposed to x-ray film (Amersham Hyperfilm ECL),following the manufacturer's instructions.

[0244] CAT-212 scFv reacted specifically with human eotaxin, as adistinct band of the predicted molecular weight was observed, while noband in the MCP-1 lane was detected. A control, irrelevant, scFv at thesame concentration did not react with either eotaxin or MCP-1.

EXAMPLE 5

[0245] Neutralisation Potency of CAT-212 in an Eotaxin-MediatedChemotaxis Assay

[0246] The potency of CAT-212 (and CAT-213, see Example 12) was testedin the chemotaxis assay, as described in Example 2. Prior to testing,monomeric scFv was prepared by FPLC gel filtration chromatography.

[0247] Preparation of Monomeric scFv

[0248] Monomeric scFv was prepared by gel-filtration of IMAC-purifiedmaterial on a Sephadex 75 column on a Pharmacia FPLC system. The columnwas run in PBS at 0.5 ml/min, and 500 μl sample was loaded. Fractionscontaining the purified monomeric scFv were collected. The concentrationof scFv was determined by reading the A_(280 nm) of the pooled peakfractions.

[0249] A typical dilution range (in assay buffer) for CAT-212FPLC-purified scFv was from 1 μg/ml down to 0.001 μg/ml.

[0250] Results

[0251] The results are shown in FIG. 3 (mean of three estimates).CAT-212 inhibited eotaxin-mediated chemotaxis of CCR3 transfected L1.2cells with an IC₅₀ of 650±83 pM. The modifications made to 3G3 hadtherefore led to over 1000-fold improvement in the potency of this scFvantibody. CAT-212 is a highly potent anti-eotaxin neutralising antibody.

EXAMPLE 6

[0252] CAT-212 Competition Assay for Eotaxin Binding to CAT-212Introduction

[0253] CAT-212 when passively immobilised to a suitable polystyrenemicrotitre plate was shown to bind eotaxin in a concentration dependentmanner as defined by its dissociation constant (K_(D)) β-emission from¹²⁵Iodine labelled eotaxin generates a detectable light signal(scintillation) when it interacts with a phosphor impregnated microtitreplate (Flash Plate™). The short range of this emission ensures that theresulting signal is due to bound antigen with little contribution fromunbound material. This technique was used to determine the K_(D) Of theeotaxin-CAT-212 interaction and the relative affinity of CAT-212 andCAT-213 (see Example 13) preparations by competitive inhibition.

[0254] Assay Protocol

[0255] The wells of a Flash Plate™ (NEN SMP200; 96-well) were coatedwith 100 μl of 40 nM IMAC-purified CAT-212 (for method of preparation,see section 2) diluted in 0.05 M carbonate-bicarbonate buffer (Sigma).The plate was sealed and incubated at 4° C. for 4 hours, then it wasemptied by inversion and blocked with 150 μl 1% Marvel in PBS (Sigma)overnight at 4° C. Immediately before use the plate was emptied byinversion and washed 3 times with PBS, then blotted dry.

[0256] The assay buffer was composed of RPMI medium (Sigma) containing0.5% bovine serum albumin (Sigma). Test samples were diluted 3-fold inbuffer to give 11 concentrations in duplicate. Fifty μl of diluted testsample was added to wells, followed by 50 μl of 30 pM [¹²⁵I]eotaxin(Amersham). The plate was sealed and incubated at room temperature for1-2 hours. The plate was counted on the Packard Topcount scintillationcounter (wells counted for 2 minutes each).

[0257] Data was analysed using a 4-parameter logistic equation usingGraphPad Prism (GraphPad Inc) to give apparent IC₅₀ values.Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogIC50−X)*HillSlope))X=logarithm of concentration, Y is the CPM.

[0258] Results

[0259]FIG. 4 shows the results of an assay in which unlabelled CAT-212,was used to compete for [¹²⁵I]eotaxin binding to CAT-212 coated to aflash plate. CAT-212 demonstrates an IC₅₀ of 42.5 pM in this assay (meanof 4 estimates).

EXAMPLE 7

[0260] Determination of CAT-212 Affinity for Eotaxin

[0261] Assay Protocol

[0262] A Flash Plate™ was coated with CAT-212 and reagents prepared asdescribed in Example 6. A serial two fold dilution of [¹²⁵I]eotaxin ±onehundred fold excess of unlabelled eotaxin in Assay Buffer was preparedand 100 μl samples in duplicate added to the coated plate. The sampleswere sealed and incubated for two hours at room temperature and counted.

[0263] The data was analysed by non-linear curve fitting using the KELLfor windows software package (Biosoft) according to the formula:Y=(B_(MAX)*[L])/(K_(D)+[L])

[0264] Where [L] is the free ligand concentration, K_(D) the affinityand B_(MAX) the maximum binding site concentration.

[0265] The data is then visualised by the use of the Scatchard Plot(FIG. 5) of the bound/free ratio vs bound giving a straight line with aslope=−1/K_(D) and an x-intercept of B_(MAX) when the ligand is bindingto a homogeneous population of binding sites.

[0266] Results

[0267] From the Scatchard plot (FIG. 5), the affinity for eotaxinbinding to CAT-212 was estimated at 146 pM.

EXAMPLE 8

[0268] Mouse Eotaxin Competition for Binding to CAT-212

[0269] Introduction

[0270] To address the in vivo activity of CAT-212/CAT-213, twostrategies were employed (see Example 14):

[0271] (1) Effecting eosinophilia in a mouse model by injection of humaneotaxin;

[0272] (2) Effecting eosinophilia in a mouse model by inducing theproduction of endogenous eotaxin.

[0273] For the second approach, it was first necessary to determinewhether CAT-212/CAT-213 recognise mouse eotaxin.

[0274] Assay Protocol

[0275] Flash Plate™ was prepared as described in Example 6. Elevenserial one in three dilutions of unlabelled human and mouse Eotaxin(R&D) in Assay Buffer and mixed with ¹²⁵I-Eotaxin prepared at anestimated concentration of 30 pM and 100 μl added to the wells induplicate. The plate was sealed and incubated at room temperature fortwo hours, counted and the data processed as described in Example 6.

[0276] Results

[0277] The IC₅₀ estimate for CAT-212 binding to mouse eotaxin is 296 nM(mean of two estimates). The data are shown in FIG. 6. CAT-212 thereforerecognises mouse eotaxin.

EXAMPLE 9

[0278] Neutralisation Potency of CAT-212 in a Calcium Flux Assay

[0279] Introduction

[0280] This functional assay is designed to measure the increase inintracellular Ca²⁺ produced when a receptor is bound and activated byits ligand. In this case, cells transfected with CCR3 are loaded with aCa²⁺ sensitive fluorescent dye (fluo-3AM) and fluorescence monitoredover time using FLIPR. In FLIPR, measurement of fluorescence in everywell is performed at programmed intervals (every 1 or 5 sec) and reagentis added simultaneously to each well. The increase in intracellular Ca²⁺concentration induced by eotaxin binding the CCR3 receptor is measuredand is proportional to the increase in fluorescence. The inhibition ofthis response caused by CAT-212 binding to and neutralising eotaxin canbe quantitated.

[0281] Assay Protocol

[0282] CCR3 cells were activated by supplementing the usual culturemedium with 0.5 μg/ml sodium butyrate (Sigma) for 24 hours prior to theexperiment to increase CCR3 expression and thus the magnitude of theresponse to eotaxin. Activated CCR3 cells were washed twice in RPMI1640and then resuspended at 4×10⁶ cells/ml in RPMI1640 containing freshlyprepared 2 μM fluo-3AM (Molecular Probes), 0.03% pluronic acid(Molecular Probes) and 0.1% FCS. The cells were then incubated for 45min at 37° C. Following this, they were washed once in RPMI1640 andtwice in FLIPR buffer (125 mM sodium chloride, 5 mM potassium chloride,1 mM magnesium chloride, 1.5 mM calcium chloride, 25 mM Hepes, 5 mMglucose and 0.1% FCS, pH 7.4). They were finally resuspended in FLIPRbuffer at 1×10⁶/ml and 100 μl plated out onto a 96-well black-walledplate with a clear base (Corning) to give 1×10⁵ cells per well. Theplate was then centrifuged at 1000 rpm for 5 min to give an even,relatively dense, monolayer of cells.

[0283] A dilution series of CAT-212 was prepared at 6×finalconcentration (final 200 nM-1.6 nM) and pre-incubated with an equalvolume of 60 nM eotaxin (Cambridge Bioscience) for 10 minutes at roomtemperature. All treatments were prepared in at least duplicate usingFLIPR buffer in a final volume of 100 μl in a 96-well polystyrene plate(Corning). The final concentration of eotaxin on the cells was 10 nM.

[0284] The plate containing the fluo-3-loaded cells was placed in FLIPRand fluorescence readings made every 1 sec for the first 60 sec then at5 sec intervals until the end. The experiment was performed at roomtemperature. For the experiment, 50 μl of each treatment was added toevery well after a 10 sec interval.

[0285] Results

[0286] The results of this experiment are illustrated in FIG. 7. It canbe seen that CAT-212 gives a dose dependent inhibition of Ca²⁺-fluxproduced by eotaxin binding its receptor on CCR3 cells. FIG. 8 shows“the area under the curve” for this data giving an approximate IC₅₀value of 5 nM.

EXAMPLE 10

[0287] Immunoreactivity of CAT-212 with Human Nasal Polyp

[0288] Introduction

[0289] Human nasal polyp is an inflammatory tissue characterised by aninfiltration of eosinophils. Eotaxin expression has been documented asbeing up-regulated in nasal polyp and eotaxin protein may be detected byimmunocytochemistry using anti-eotaxin antibodies (Ponath et al.,1996a). CAT-212 was therefore screened for immunoreactivity withsections of human nasal polyp.

[0290] Preparation of Tissues for ICC

[0291] Human nasal polyp tissue was obtained from surgical samples.Tissues were cut into 5 mm³ chunks and mounted onto cork pieces using adrop of optimum cutting tissue compound (OCT; Sakura). To freeze thetissues, 20 mls of isopentane was cooled in a bath of liquid nitrogenand the mounted tissues immersed for 30 seconds. The frozen tissues werethen placed into a cryotube and immersed in liquid nitrogen for afurther 30 seconds. Tissue blocks were stored frozen at B70° C. To cutsections, OCT compound was applied to a cryostat chuck and the frozentissue embedded. The chuck and tissue were then snap frozen for 30seconds in liquid nitrogen. The tissue was then mounted onto a cryostatand 5 micron cryosections of each human tissue cut onto microscopeslides.

[0292] Preparation of Phage Antibodies for ICC

[0293] Phage antibody clones were rescued as described in Example 1.Phage-containing supernatant was preblocked with 1% BSA before use inICC.

[0294] Protocol

[0295] Human tissue sections were fixed by immersion in acetone atambient temperature for 15 minutes, air dried and then washed threetimes in PBSt for 10 minutes (total). The test scFvs were diluted inPBST and applied to the sections for 2 hours at room temperature. Slideswere washed 3 times in PBST and incubated with mouse 9E10 antibodydiluted {fraction (1/100)} in PBST. Sections were washed 3 times in PBSTand incubated in EnVision anti-mouse peroxidase polymer as supplied(DAKO K4006) for 30 minutes. Sections were again washed 3 times in PBSTand stained with 3-amino-9-ethyl-carbazole peroxidase substrate (AEC;Sigma). AEC substrate was prepared by diluting a stock solution (2.4mg/ml of AEC dissolved in dimethylformamide) 1:10 in 20 mM sodiumacetate buffer, pH 5.2 and adding 0.15% (v/v) of hydrogen peroxide. Onehundred μl of AEC substrate solution was added to each section andincubated for 5-10 minutes followed by washing in tap water containing0.1% Tween to stop colour development. The slides were thencounterstained with haematoxylin (DAKO S2020) for <5 seconds and thenwashed 3 times in water. Washed sections were then coated in aqueousmount and a glass cover slip applied.

[0296] Results

[0297] CAT-212 at 30 μg/ml stained sections of human nasal polyp in anequivalent manner to that seen with a positive control anti-eotaxinantibody (Cambridge Bioscience) at 50 μg/ml. There was no detectablestaining in the substrate only control or with an irrelevant scFv at thesame concentration. CAT-212 demonstrated specific cytoplasmic stainingin the covering epithelium, and in elongated mononuclear cells andendothelium situated in the stroma of nasal polyp. This is consistentwith the published ICC profile obtained with an anti-eotaxin antibody onnasal polyp (Ponath et al., 1996a). To confirm that the staining wasspecific for eotaxin, a competition ICC was undertaken in which 40 μg/mlCAT-212 was pre-bound to 133 μg/ml, 660 μg/ml and 1.33 mg/ml eotaxinprior to addition to the nasal polyp sections. CAT-212 alone gave goodstaining as described above. When CAT-212 was pre-bound to eotaxin,there was a dose-dependent inhibition of binding to nasal polyp with1.33 mg/ml eotaxin virtually abolishing all specific staining.

EXAMPLE 11

[0298] Conversion of CAT-212 to IgG4 Format (CAT-213)

[0299] Introduction

[0300] The vectors used in the conversion of the scFv (CAT-212) intowhole antibody (IgG4; CAT-213) format were as follows: pGamma4 (V_(H)expression vector) and pMR15.1 (V_(L) expression vector). Both of thesevectors were obtained from Lonza Biologics.

[0301] Protocol

[0302] All primers used are referenced to SEQ ID NO.'s in Table 3. TheV_(H) DNA and V_(K) DNA were initially amplified up usingoligonucleotides p113/p132 and p109a/p110b respectively. All the PCRreactions used PwoI polymerase (Roche) for its proof readingcapabilities and PCR conditions were 25 cycles of 94° C., 30 seconds;50° C., 30 seconds; 72° C., 60 seconds. The signal sequence for bothV_(N) and V_(K) were added on by amplification with p10/p132 andp11/p110b respectively. The V_(H) and V_(K) fragments of DNA weredigested in parallel with their acceptor vectors, with HindIII, ApaI andBstBI, BsiWI respectively. Fragments were then ligated together, thusconstructing the separate plasmids. Both plasmids were digested withBamHI and Not I and ligated together to form the final construct. Allthe restriction digests and ligations were performed using enzymes fromNew England Biolabs, using the buffers recommended by the supplier.

[0303] For transformation of the vectors, electrocompetent E. coli cells(DH5α) were used. The electroporation was carried out in 0.2 cm gapelectroporation cuvettes, into which 5 μl of ligated DNA was added to100 μl of E. coli cells. The cells were given a single pulse of 2.5 kVat 200 Ω followed by a 20 minute recovery period in 2TY at 37° C. in ashaking incubator. Aliquots were plated out onto 2TYAG agar plates andincubated at 37° C. overnight. The following day the colonies werepicked and screened by PCR using Taq polymerase and the appropriateprimers.

[0304] A clone with a correctly-sized insert was grown in 100 ml 2TY(containing 100 μg/ml ampicillin) overnight at 37° C. in a shakingincubator. The cells were harvested by centrifugation at 3,000 g for 15minutes, and a QIAGEN maxi-prep kit was used to extract the plasmid DNA.The DNA concentration was determined spectrophotometrically assumingthat an A_(260 nm) of 1=50 μg/ml. The final construct was sequenced withprimers p24, p34 and p36 and p37 to confirm the correct sequence.

[0305] A 36 plate transfection was performed in NSO cells byelectroporation (250V) using the gs system (Lonza) using the glutaminesynthetase gene as the selectable marker. Wild type NSO cells were grownin DMEM (Sigma) containing 10% dialysed FCS with 2 mM glutamine. 6×10⁷NSO cells were transfected with 300 μg of DNA, linearised by Pvu I.After electroporation the cells were resuspended in DMEM with glutamineand plated out into 36×96-well plates (50 μl/well) and incubated at 37□C. in 5% CO₂. The following day, 150 μl/well of selective medium (DMEMwithout glutamine) was added. After approximately 3 weeks the colonieswere screened by ELISA (see below) using an irrelevant antibody as anegative control. All colonies producing >20 μg/ml were expanded into24-well plates and then into duplicate T25 flasks. One flask was grownto saturation and the other frozen down. The first cell line (notclonal) named 4B7 was adapted to serum free medium and expanded to 2 Lvolume for purification using protein A.

[0306] Screening ELISA Assay to Detect IgG Expression

[0307] Each well of an ELISA plate (Immulon 4, Dynex technologies) wascoated with 100 μl of 1 μg/ml goat anti-human IgG (Harlan) in 50 mMsodium bicarbonate/carbonate buffer, pH 9.6, at 4□ C. overnight. Plateswere washed 3 times in PBS containing 0.05% (v/v) Tween 20 (PBST/0.05).CAT-213 was diluted in PBST/0.05 and a series of 2-fold dilutions weregenerated across the plate. The plate was incubated for 1 hour at roomtemperature, and then washed 3 times in PBST/0.05. One hundred 1 of1:5000 HRP conjugated goat anti-human IgG antibody (Harlan), diluted inPBST/0.05 was added to each well and incubated for 30 minutes at roomtemperature. The plate was washed 3 times in PBST/0.05. Following this,100 μl freshly prepared HRP substrate buffer (0.4 mg/mlo-phenylenediamine in 24 mM citric acid, 52 mM sodium hydrogenphosphate, pH 5.2, containing 5 l H₂O₂/50 ml buffer added just beforeuse) was added to each well. After 5-10 minutes the reactions werestopped with the addition of 50 μl 12.5% sulphuric acid. The A₄₉₀ wasmeasured.

[0308] The cell line initially expressing the highest amount of IgG, asindicated by a high signal in the screening ELISA, was chosen forexpansion. This entailed expanding it from a 96 well plate up to a T75flask. This cell line was then adapted to a serum free medium (LonzaNM2) involving serial dilutions, decreasing the serum by half every time(starting percentage of serum is 10%). Once adapted to serum freemedium, the cell line was then grown to saturation in 150 ml flasks, andharvested at less than 10% cell viability. The supernatant was clarifiedby centrifugation then filtered through a 0.22 μm filter. The antibodywas purified from the supernatant using a Protein A affinity column.

[0309] Binding Assay to Detect Anti-Eotaxin Specific IgG

[0310] Each well of an ELISA plate (Immulon 4, Dynex technologies) wascoated with 100 μl 0.5 μg/ml eotaxin (Albachem) in 50 mM sodiumbicarbonate/carbonate buffer, pH 9.6, at 4° C. overnight. The rest ofthe protocol is the same for the screening assay (see section above).Results, including an irrelevant control antibody, are shown in FIG. 9.

EXAMPLE 12

[0311] Neutralisation Potency of CAT-213 in an Eotaxin-Mediated

[0312] Chemotaxis Assay

[0313] Assay Protocol

[0314] The potency of CAT-213 was tested in the chemotaxis assay,essentially as described in Example 2. Prior to testing, CAT-213 waspurified from the supernatant using a Protein A affinity column (as inExample 11). A typical dilution range for Protein A purified CAT-213 wasfrom 100 nM down to 0.03 nM in assay buffer.

[0315] Results

[0316] The results of this assay are shown in FIG. 3. CAT-213 inhibitedeotaxin-mediated chemotaxis of CCR3 transfected L1.2 cells with an IC₅₀of 700+350 pM. The potency is similar to that seen for CAT-212 in thisassay.

EXAMPLE 13

[0317] CAT-213 Competition Assay for Eotaxin Binding to CAT-212

[0318] Assay Protocol

[0319] The Flash Plate assay was performed essentially as described inExample 6. The Flash Plate was coated with 40 nM CAT-212. CAT-212,CAT-213, and eotaxin were diluted in assay buffer and 50 l added to thewells followed by 50 l of 60 pM [¹²⁵I] eotaxin.

[0320] Results

[0321] The IC₅₀ of CAT-213 in this assay is 59.3 pM (mean of 4estimates, FIG. 4). This is similar to the value obtained for the scFv,CAT-212.

EXAMPLE 14

[0322] Effects of CAT-212 and CAT-213 in an in vivo Model of AllergicInflammation

[0323] The air pouch model of allergic inflammation was chosen as aconvenient model in which to study eosinophilia. Dexamathasone (asteroidal anti-inflammatory drug) has been shown to block eosinophilrecruitment to the air pouch in this model (see Das et al., 1997). Otheranti-eotaxin antibodies have previously been shown to block eosinophilrecruitment in a range of in vivo models (discussed already above) inwhich eosinophilia was stimulated with either local administration ofeotaxin or induced by ovalbumin (antigen) challenge in ovalbuminsensitized animals.

[0324] The effects of CAT-212 or CAT-213 were investigated in the airpouch model in ovalbumin sensitized mice in which eosinophilia wasinduced by either recombinant human eotaxin or ovalbumin administeredintra-air pouch (i.po.).

[0325] Methods

[0326] Female Balb/c mice (17B21 g, supplied by Harlan U. K. or CharlesRiver) were housed within the Small Animal Barrier Unit at BabrahamInstitute (Cambridgeshire). Mice was housed 3 to a cage with a 12 hday/night cycle (lights on 7 am). Animals were allowed to acclimatize tothe animal house for at least 2 weeks prior to experimentation andallowed food and water ad libitum.

[0327] Sensitization was carried out as reported by Das et al., 1997.Briefly, mice were sensitized to ovalbumin by subcutaneous (s.c.)injection of 100 g ovalbumin in aluminium hydroxide gel (0.4 ml of asaline containing 3.3 mg aluminium hydroxide; Rehydragel) on days 1 and8. An air pouch was formed on the back of the mice in the mannerpreviously reported by Das et al., 1997. On day 9, mice wereanaesthetised with isoflurane and 2.5 ml sterile air (0.25 m filtered)injected s.c. on the back of each mouse. On day 12, mice were injectedwith a further 2.5 ml sterile air to re-inflate the air pouch. On day 15mice were challenged with either eotaxin or ovalbumin.

[0328] Human Eotaxin Challenge Procedure

[0329] In a pilot experiment it was established that human recombinanteotaxin (Albachem) administered i.po. could evoke eosinophilia inovalbumin sensitized mice. 30 min before administration of humaneotaxin, recombinant murine IL-5 (IL-5; 100 pmol kg⁻¹) was injectedintravenously (i.v.) to increase the circulating pool of eosinophils(see Collins et al., 1995). After 6 h, human eotaxin (1 μg, 0.5 mli.po.) caused a significant increase in eosinophil recruitment to theair pouch compared to saline (0.5 ml i.po.; eotaxin 5.3 . 1.1, saline1.2 . 0.3 eosinophils×10⁵; n=6, P<0.01 Mann-Whitney test). In furtherexperiments 100 pmols kg⁻¹ IL-5 (i.v.) as well as 1 μg human eotaxin(i.po.) were used and measurements were made 6 h after administration ofeotaxin. A control group treated with saline alone was also included inall experiments to give the baseline cell influx (sham challenge group).

[0330] Ovalbumin Challenge Procedure

[0331] The challenge dose of ovalbumin was determined from a series ofpilot experiments in ovalbumin sensitized mice. In an initialexperiment, Ovalbumin 1 and 10 but not 100 μg (0.5 ml, i.po.) was shownto cause a significant eosinophil recruitment compared with vehicletreated animals (0.5 ml saline, i.po.) after 24 h. As 10 μg Ovalbuminproduced the most reproducible response (ovalbumin 6.4±1.1, saline1.46±0.23 eosinophils×10⁵; n=6, P<0.01, ANOVA with Dunnett's Test) thisdose was subsequently used in a time course experiment. In thisexperiment-mice received either saline or 10 μg ovalbumin (i.po.) andcell influx was assessed between 2-72 h after challenge. The 6 h timepoint was selected as eosinophil (as well as total cell) influx to theair pouch was maximal at this time (ovalbumin 5.4±1.4, saline 1.1±0.2eosinophils×10⁵; n=5-6). In all subsequent experiments 10 μg ovalbumin(i.po.) was administered and measurements taken at 6 h. A control groupchallenged with saline alone was also included in all experiments togive the baseline cell influx (sham challenge group). Furthermore, mouseeotaxin levels were shown to be elevated following ovalbumin challenge(10 μg i.po.) in ovalbumin sensitized animals (saline control 93±12,ovalbumin challenge 3539±372 pg ml⁻¹; n=8-9, P<0.001, Mann-Whitneytest).

[0332] Drug Administration

[0333] Systemic IgG antibody treatments (CAT-213, CAT-001 [null IgGcontrol], or anti-mouse eotaxin IgG1 [R&D Systems]) were i.v. 30 minbefore administration of human eotaxin or ovalbumin. Local intra-airpouch treatments with CAT-212, CAT-171 [null scFv control] or CAT-213were concomitant with administration of human eotaxin or ovalbumin.Vehicle control groups were included in all antibody experiments andthese mice received PBS. All i.v. injections were made via the tail veinin a volume of 100 μl and all i.po. injections were made in a totalvolume of 0.5 ml.

[0334] Quantification of Results

[0335] Mice were killed prior to lavage by either CO₂ asphyxiation or,when plasma samples were required, an overdose of sodium pentobarbitonewas followed by cardiac puncture. The air pouches were lavaged with icecold PBS (1 ml; without calcium or magnesium; Sigma) containing 5 U ml⁻¹heparin and stored on ice. An aliquot (10 μl) of lavage fluid wasremoved for assessment of total cells (fast-read, disposable cellcounter, Immune Systems Ltd) and a further sample containing 100,000cells was taken for cytospin preparation. The remaining lavage fluid wascentrifuged 1000 g for 5 min, the supernatant was then aliquoted andstored at B70° C. The cell pellet was resuspended and cytospinsprepared, air dried and stained with Wrights stain for differential cellcounting. In some instances the lavage supernatant was assayed foreotaxin by ELISA (R&D Systems).

[0336] All results shown are mean ±SE. The cell influx data areexpressed as the number of cell per air pouch or, for drug treatment, as% inhibition of eosinophil influx. % inhibition of eosinophil influx wascalculated from cell number data for each treatment group with thefollowing equation:$\frac{( {{{Mean}\quad {cell}\quad {influx}\quad {{challenged}\quad\lbrack {{PBS}\quad {control}} \rbrack}\quad {group}}\quad - {{Cell}\quad {influx}\quad {test}\quad {mouse}}} )}{( {{Mean}\quad {cell}\quad {influx}\quad {{challenged}\quad\lbrack {{PBS}\quad {control}} \rbrack}\quad {group}\quad B\quad {Mean}\quad {cell}\quad {influx}\quad {sham}\quad {challenged}\quad {group}} )} \times 100$

[0337] The mean ±SE was then calculated for each treatment group.

[0338] Most raw data were statistically analyzed with either ANOVA withDunnett's. Students unpaired t test or Mann-Whitney tests were alsoused. All statistical analysis was performed using the Instat softwareand differences between mean values were taken as significant whenP<0.05.

[0339] Results

[0340] Effect of CAT-212 or CAT-213 in Mice Treated with Eotaxin i.po.

[0341] CAT-212 (0.0005, 0.005 & 0.05 mg kg⁻¹ i.po.) significantlyattenuated the eosinophilia (32, 79 and 95% inhibition, respectively;n=8) caused by human eotaxin (1 g; i.po) in IL-5 treated, ovalbuminsensitized mice (FIG. 10). CAT-171 null control scFv (0.05 g kg⁻¹ i.po.)had no effect on eosinophil recruitment (−5 . 15% inhibition; n=8).

[0342] CAT-213 (0.001, 0.01, 0.1 & 1 mg kg⁻¹, n=7-8) administered i.po.concurrently with human eotaxin (1 μg, i.po) caused a potentdoseBrelated inhibition of eosinophilia (FIG. 10).

[0343] All doses of CAT-213 were effective and maximum inhibition of 94%was observed with CAT-213 at 1 μg kg⁻¹. CAT-001 1 μg kg⁻¹ had littleeffect on cell influx (7 . 1% inhibition, n=8).

[0344] CAT-213 (0.01, 0.1, 1 & 10 mg kg⁻¹) administered i.v. 30 minbefore i.po. injection of human eotaxin (1 μg) caused a significantdose-dependent inhibition (46, 73, 79 & 91%, respectively, n=8) ofeosinophil recruitment in IL-5 treated, ovalbumin sensitised mice (FIG.10). CAT-001 control IgG4 (10 mg kg⁻¹) did not significantly affecteosinophil recruitment (8±1% inhibition, n=8).

[0345] Thus, intra-air pouch administration of CAT-213 is equipotentwith CAT-212 in its ability prevent eosinophilia in vivo (ED₉₀ for bothCAT-213 and CAT-212 is approximately 1×10⁻⁹ mols kg⁻¹) CAT-213 andCAT-212 block eosinophilia in vivo by neutralising human eotaxin; theseantibodies have previously been shown to have a similar neutralisingpotency against human eotaxin when compared in the in vitro chemotaxisassay (see Example 12). CAT-213 also prevents eosinophil recruitmentfollowing i.v. administration (CAT-212 was not tested i.v.). However,CAT-213 is a more potent inhibitor of human eotaxin induced eosinophiliawhen given locally (CAT-213 i.po. is approximately 10 fold more potentcompared with i.v. administration; FIG. 10).

[0346] Effect of CAT-213 in Ovalbumin i.po. Treated Animals

[0347] CAT-213 (0.01, 0.1, 1 & 10 mg kg⁻¹, n=7B8) i.po. dose-dependentlyinhibited eosinophil recruitment induced by ovalbumin. CAT-213 andovalbumin were administered concurrently. CAT-213 0.1 mg kg⁻¹ and 10 mgkg⁻¹ caused 60% and 97% inhibition, respectively. CAT-213 i.po. alsoinhibited mononuclear cell influx to the air pouch, however, neutrophilrecruitment was not significantly affected (Table 2). CAT-001 10 mg kg⁻¹i.po. had little effect on cell influx (inhibition of eosinophil influxwas B20±18%, n=8).

[0348] CAT-213 administered i.v. significantly inhibited eosinophilrecruitment induced by ovalbumin (i.po.). CAT-213 (0.01, 0.1, 1 & 10 mgkg⁻¹ i.v., n=8) given 30 min before ovalbumin blocked eosinophilia at 6h. 1 and 10 mg kg⁻¹ caused a significant inhibition of eosinophilrecruitment by 56 and 85%, respectively. CAT-213 0.01 and 0.1 mg kg⁻¹ aswell as CAT-001 (null antibody control) 10 mg kg⁻¹ were inactive (FIG.19; CAT-001 inhibition 14 . 14%, n=8). CAT-213 i.v. (but not CAT-001)caused a dose-related inhibition of ovalbumin induced neutrophil andmononuclear cell recruitment to the air-pouch (Table 2).

[0349] Again, the data above provides indication that CAT-213 is a morepotent inhibitor of eosinophilia when given locally to the air pouch(approximately 10 fold more potent than i.v. administration; FIG. 19).However, systemic (i.v.), but not local (i.po.) administration ofCAT-213 has the ability to block neutrophil influx. Both systemic andlocal administration of CAT-213 can inhibit mononuclear cell chemotaxis.

[0350] In a separate experiment, the effects of systemic (i.v.)administration of anti-mouse eotaxin IgG2A (R&D Systems) wereinvestigated in ovalbumin (i.po.) challenged mice to provide comparativedata. Anti-mouse eotaxin (0.05, 0.5 & 5 mg kg⁻¹, n=7-8) caused adose-related inhibition of eosinophil recruitment (14, 37 & 67%,respectively). Thus CAT-213 and the anti-mouse eotaxin IgG2A produced asimilar inhibition of eosinophil chemotaxis both in magnitude ofresponse and potency (FIG. 19).

SUMMARY

[0351] In summary, the human anti-eotaxin antibodies, CAT-212 (scFv) andCAT-213 (IgG4) potently block eosinophilia in an in vivo model ofallergic inflammation. CAT-213 is effective when given both locally andsystemically. Systemic administration of CAT-213 has the additionalaction of blocking neutrophil as well as mononuclear cell chemotaxis.These data are consistent with an action of CAT-213 and CAT-212 inblocking the biological response to eotaxin in vivo.

EXAMPLE 15

[0352] Neutralisation Potency of CAT-213 in an Eotaxin-MediatedChemotaxis Assay Using L1.2 CCR-3 Transfected Cells: Rhesus Monkey andMouse Eotaxin

[0353] The ability of CAT-213 to neutralise rhesus monkey and mouseeotaxin was assessed in the chemotaxis assay, essentially as describedin Example 2.

[0354] Assay Protocol

[0355] The assay protocol followed was essentially the same as thatdescribed for Example 2. Chemotaxis was induced with either rhesusmonkey eotaxin or murine eotaxin. Rhesus monkey (125 ng/ml) or murine(50 ng/ml) eotaxin were incubated in the lower chamber of a Transwellculture system prior to the addition of 1×10⁶ or 2×10⁶ L1.2-CCR3 cells,respectively to the upper chamber of the Transwell. Migrated cells werequantitated by FACS analysis and were counted at a high flow rate for 1minute.

[0356] Results

[0357] CAT-213 inhibited rhesus monkey and mouse eotaxin-mediatedchemotaxis of L1.2-CCR3 cells with geometric mean IC₅₀ and 95%confidence interval values of 3.03×10⁻⁷M (1.01×10⁻⁷, 9.0×10⁻⁷M) and2.63×10⁻⁶M (1.30×10⁻⁶, 5.34×10⁻⁶M), respectively (FIG. 12). The datarepresent at least 3 experiments performed in triplicate and duplicate,respectively.

EXAMPLE 16

[0358] Neutralisation Potency of CAT-213 in an Eotaxin-MediatedChemotaxis Assay Using Human Eosinophils

[0359] The chemotaxis assay is a relevant in vitro assay system as itassesses the ability of eotaxin to chemoattract cells expressing CCR3.These experiments are of increased physiological relevance as in thiscase the CCR3-expressing cells are human eosinophils obtained directlyfrom blood.

[0360] Preparation of Eosinophils

[0361] Eosinophils were isolated from heparinized peripheral blood ofatopic, non-asthmatic donors with no symptoms of allergic disease. Bloodwas mixed with ⅕ volume dextran solution (6% w/v in saline) anderythrocytes were allowed to sediment for 45 minutes at roomtemperature. The erythrocyte-depleted plasma was layered ontoLymphoprepe® and centrifuged at 800 g for 25 minutes at 20° C. toseparate mononuclear cells from granulocytes.

[0362] Granulocyte pellets were depleted of residual erythrocytes viatwo rounds of hypotonic lysis, after which neutrophils were labelledwith anti-CD16-coated superparamagnetic microbeads (MACS, MiltenyiBiotec; 2 μl beads per 3×10⁶ granulocytes) by a 30-minute incubation at0° C. The cells were then loaded onto a 10 ml steel wool column in astrong magnetic field and the unlabelled cells eluted with four columnvolumes of ice-cold labelling buffer (PBS, 2×10⁻³M EDTA, 0.5% w/v bovineserum albumin). The eluted cells were routinely >95% eosinophils.

[0363] Assay Protocol

[0364] Eosinophils were resuspended in HBSS supplemented with 1×10⁻²MHEPES and 0.3% w/v BSA, and incubated for at least 45 min at 37° C./5%CO₂ prior to use in chemotaxis assays. Samples (medium or 1×10⁻⁸M humaneotaxin, plus or minus CAT-213 or CAT-001 (control antibody)) were addedto the lower wells of 48-well microchemotaxis chambers.Polyvinylpyrrolidone (PVP)-free polycarbonate membrane filters (8-μmpore size) were placed between the lower and upper wells and thechambers were incubated for 30 minutes at 37° C./5% CO₂. Cells were thenadded to the upper wells and the chambers incubated for a further 60minutes at 37° C./5%CO₂. At the end of this period, non-migrating cellswere scraped from the upper surface of the filters; the filters weredried, fixed in methanol and stained using May-Grunwald/Giemsa. Migratedcells were counted in 5 high-power (×400) fields for each sample.

[0365] Results

[0366] The results of three separate experiments performed withtriplicate points are shown in FIG. 13 (mean ±SEM). CAT-213 inhibitedeotaxin-mediated chemotaxis of human eosinophils with a geometric meanIC₅₀ and 95% confidence intervals of 1.53×10⁻⁹M (3.09×10⁻¹¹,7.54×10⁻⁸M). CAT-001 (the control antibody) did not affect chemotaxis.Therefore, CAT-213 neutralises eotaxin-induced chemotaxis of humaneosinophils.

EXAMPLE 17

[0367] Neutralisation Potency of CAT-213 in an Eotaxin-MediatedEosinophil Shape Change Assay

[0368] Shape change is a requisite process in chemotaxis and can betaken as evidence of a migratory response, to come. In order foreosinophils to move from the circulation into the tissues in response toan eotaxin gradient (chemotaxis), reorganisation of cytoskeletalelements must occur. This reorganisation is associated with specificchanges in cell morphology which can be visualised on a flow cytometeras changes in forward scatter (FSC). The assay used in this example usesa method described by Sabroe et al (1999).

[0369] Assay Protocol

[0370] Experiments were performed using granulocyte preparations whichwere obtained as follows. Peripheral blood was taken into syringescontaining EDTA (150 μl 0.5M EDTA per 10 ml blood) and the erythrocytessedimented by addition of 1 ml of 6% dextran-T500 in 0.9% saline per 10ml of blood. Sedimentation was allowed to occur for 40 minutes at roomtemperature. The erythrocyte-depleted plasma was then layered onto adiscontinuous 70%-80% percoll gradient and centrifuged at 1137 g for 20minutes at room temperature to separate mononuclear cells fromgranulocytes. The resultant granulocyte layer was washed in PBS and thenpelleted at 306 g for 5 minutes at room temperature. The cell pellet wasthen resuspended in shape change buffer (PBS, 1×10⁻³M CaCl₂, 1×10⁻³MMgCl₂, 1×10⁻²M HEPES, 1×10 ⁻²M glucose, 0.1% BSA, pH 7.3). Cells werethen incubated at 37° C. for 30 min.

[0371] 3×10⁻⁹M eotaxin or buffer, plus or minus CAT-213 or CAT-001(control) were added to polypropylene flow cytometer tubes in a volumeof 300 μl. 5×10⁵ cells (in 100 μl) were added to each tube. Tubes werethen immediately incubated at 37° C. for 7 minutes before beingtransferred to an ice water bath. Finally, 25 μl Cellfix buffer (10×)was added and individual tubes were read on a flow cytometer.Eosinophils were identified by their autofluorescence in the FL2channel. FSC was then assessed.

[0372] Results

[0373] The results of this assay are shown in FIG. 14. CAT-213 inhibitedshape change evoked in human eosinophils by human eotaxin, with ageometric mean IC₅₀ and 95% confidence interval of 7.14×¹⁰M (2.07×10¹⁰,2.44×10⁻⁹M). CAT-001 did not affect eotaxin induced shape change. Dataare expressed as mean ±SEM from 5 experiments performed with duplicatepoints, with cells from separate donors.

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[0421] TABLE 1 Coating concentration Test Antigen (μg/ml) Supplier HumanEotaxin 10 Cambridge Bioscience Mouse Eotaxin 10 Cambridge BioscienceMIP-1α 10 Cambridge Bioscience MCP-1 10 Cambridge Bioscience MCP-2 10Cambridge Bioscience MCP-3 10 Cambridge Bioscience MCP-4 10 CambridgeBioscience IL-1α 1 Cambridge Bioscience IL-1β 1 Cambridge BioscienceIL-5 1 R&D Systems IL-12 1 Gift: from Genetics Institute IL-18 1 R&DSystems TGF-β1 0.5 ImmunoKontact TGF-β2 0.5 ImmunoKontact TNFα 10 Gift:BASF Bio-Research Corporation RANTES 10 Peprotech

[0422] TABLE 2 The Effect of CAT-213 i.v. on Ovalbumin-inducedNeutrophil and Mononuclear Cell Recruitment in Ovalbumin Sensitized MiceDose Mononuclear (mg kg⁻¹ Neutrophils cells Treatment i.v.) (%Inhibition) (% Inhibition) n CAT-213 0.01 9 ± 13 −7 ± 18 8 0.1 37 ± 9 49 ± 23 8 1 41 ± 12* 101 ± 30* 8 10 61 ± 6**  156 ± 12** 8 CAT-001 10 1± 10 −3 ± 57 8

[0423] Mean ±SE % inhibition values for the effect of CAT-213 or CAT-001on neutrophil or mononuclear cell chemotaxis. The effect of antibodytreatment was statistically evaluated by performing one way ANOVA withDunnett's test using the differential cell count data. *P<0.05, **P<0.01compared to ovalbumin challenged PBS control animals. CAT-213 i.v.significantly inhibited neutrophil and mononuclear cell chemotaxis.TABLE 3 List of primers used for conversion of CAT-212 to IgG format SEQID NO. Primer 11 P10 12 P11 13 P24 14 P34 15 P36 16 P37 17 P109a 18P110b 19 P113 20 P132

[0424]

1 20 1 354 DNA homo sapiens 1 caggtgcagc tggtgcaatc tgggggaggcgtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagtagctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagttatatcatatg atggaagcat taaacattat 180 gcagactccg tgaagggccg attcaccatctccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag aactgacgacacggctgtat attactgtgc gggagatacg 300 gactacgggg acatcgaccc gtggggtcagggcaccatgg tgacggtctc gagt 354 2 118 PRT homo sapiens 2 Gln Val Gln LeuVal Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met HisTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val IleSer Tyr Asp Gly Ser Ile Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly ArgPhe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu GlnMet Asn Ser Leu Arg Thr Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala GlyAsp Thr Asp Tyr Gly Asp Ile Asp Pro Trp Gly Gln Gly Thr 100 105 110 MetVal Thr Val Ser Ser 115 3 326 DNA homo sapiens 3 acatccagat gacccagtctccatcttccg tgtctgcatc tgtaggagac agagtcacca 60 tcacttgtcg ggcgagtcaggatattagca gctggttagc ctggtatcag cagaaacctg 120 ggaaagcccc taagctcctgatctatgctg catccagttt gcaaagtggg gtcccatcaa 180 ggttcagcgg cagtggatctgggacagatt tcactctcac catcagcagc ctgcagcctg 240 aagattttgc aacttactattgtcagcagg ctagcagttt cccctcgatc accttcggcc 300 aagggacacg actggagattaaacgt 326 4 109 PRT homo sapiens 4 Asp Ile Gln Met Thr Gln Ser Pro SerSer Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys ArgAla Ser Gln Asp Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln SerGly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr TyrCys Gln Gln Ala Ser Ser Phe Pro Ser 85 90 95 Ile Thr Phe Gly Gln Gly ThrArg Leu Glu Ile Lys Arg 100 105 5 5 PRT homo sapiens 5 Ser Tyr Gly MetHis 1 5 6 17 PRT homo sapiens 6 Val Ile Ser Tyr Asp Gly Ser Ile Lys HisTyr Ala Asp Ser Val Lys 1 5 10 15 Gly 7 9 PRT homo sapiens 7 Asp Thr AspTyr Gly Asp Ile Asp Pro 1 5 8 11 PRT homo sapiens 8 Arg Ala Ser Gln AspIle Ser Ser Trp Leu Ala 1 5 10 9 7 PRT homo sapiens 9 Ala Ala Ser SerLeu Gln Ser 1 5 10 10 PRT homo sapiens 10 Gln Gln Ala Ser Ser Phe ProSer Ile Thr 1 5 10 11 30 DNA Artificial Sequence Primer 11 ctaagcttactgagcacaca ggacctcacc 30 12 35 DNA Artificial Sequence Primer 12aattttcgaa ctacagttac tgagcacaca ggacc 35 13 21 DNA Artificial SequencePrimer 13 ggaggtgctc ctggagcagg g 21 14 29 DNA Artificial SequencePrimer 14 ctgttccttt ccatgggtct tttctgcag 29 15 27 DNA ArtificialSequence Primer 15 ttccatgggt cttttctgca gtcaccg 27 16 27 DNA ArtificialSequence Primer 16 tatggctgat taatgatcaa tgaattc 27 17 45 DNA ArtificialSequence Primer 17 tttggatatc tctccacagg tgtccactcg gacatccaga tgacc 4518 37 DNA Artificial Sequence Primer 18 cggccaaggg acacgactgg agattaaacgtacggta 37 19 54 DNA Artificial Sequence Primer 19 tttggatatc tctccacaggtgtccactcc caggtgcagc tggtgcaatc tggg 54 20 46 DNA Artificial SequencePrimer 20 atgggccctt ggtggaagca ctcgagaccg tcaccatggt gccctg 46

1. A specific binding member that binds human eotaxin and whichcomprises an antibody VH domain selected from the group consisting ofthe CAT-212 VH domain (SEQ ID NO. 2), and a VH domain comprising one ormore VH CDR's with an amino acid sequence selected from SEQ ID NO. 5,SEQ ID NO. 6 and SEQ ID NO. 7; and/or an antibody VL domain selectedfrom the group consisting of the CAT-212 VL domain (SEQ ID NO. 4), and aVL domain comprising one or more VL CDR∝s with an amino acid sequenceselected from SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO.
 10. 2. Aspecific binding member according to claim 1 comprising an antibody VHdomain comprising the VH CDR∝s with the amino acid sequences of SEQ IDNO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, which specific binding membercompetes for binding to eotaxin with an eotaxin-binding domain of anantibody comprising the CAT-212 VH domain (SEQ ID NO. 2) and the CAT-212VL domain (SEQ ID NO. 4).
 3. A specific binding member according toclaim 1 or claim 2 comprising the CAT-212 VH domain (SEQ ID NO. 2).
 4. Aspecific binding member according to claim 3 comprising the CAT-212 VLdomain (SEQ ID NO. 4)
 5. A specific binding member according to any oneof claims 1 to 3 that binds eotaxin with affinity equal to or betterthan the affinity of an eotaxin antigen-binding site formed by theCAT-212 VH domain (SEQ ID NO. 2) and the CAT-212 VL domain (SEQ ID NO.4), the affinity of the specific binding member and the affinity of theantigen-binding site being as determined under the same conditions.
 6. Aspecific binding member according to any one of claims 1 to 3 thatneutralises eotaxin.
 7. A specific binding member according to claim 6that neutralises eotaxin with a potency equal to or better than thepotency of an eotaxin antigen-binding site formed by the CAT-212 VHdomain (SEQ ID NO. 2) and the CAT-212 VL domain (SEQ ID NO. 4), thepotency of the specific binding member and the potency of theantigen-binding site being as determined under the same conditions.
 8. Aspecific binding member according to any one of claims 1 to 7 thatcomprises an scFv antibody molecule.
 9. A specific binding memberaccording to any one of claims 1 to 7 that comprises an antibodyconstant region.
 10. A specific binding member according to claim 9 thatcomprises a whole antibody.
 11. A specific binding member according toclaim 9 or claim 10 comprising an IgG4 constant region.
 12. An isolatednucleic acid which comprises a nucleotide sequence encoding a specificbinding member or antibody VH or VL domain of a specific binding memberaccording to any one of claims 1 to
 11. 13. A host cell transformed withnucleic acid according to claim
 12. 14. A method of producing a specificbinding member or antibody VH or VL domain, the method comprisingculturing host cells according to claim 13 under conditions forproduction of said specific binding member or antibody VH or VL domain.15. A method according to claim 14 further comprising isolating and/orpurifying said specific binding member or antibody VH or VL variabledomain.
 16. A method according to claim 14 or claim 15 furthercomprising formulating the specific binding member or antibody VH or VLvariable domain into a composition including at least one additionalcomponent.
 17. A method of obtaining a specific binding member thatbinds eotaxin, the method comprising providing by way of addition,deletion, substitution or insertion of one or more amino acids in theamino acid sequence of the CAT-212 VH domain (SEQ ID NO. 2) one or moreVH domains each of which is an amino acid sequence variant of theCAT-212 VH domain, optionally combining one or more VH domain amino acidsequence variants thus provided with one or more VL domains to provideone or more VH/VL combinations; and/or providing by way of addition,deletion, substitution or insertion of one or more amino acids in theamino acid sequence of the CAT-212 VL domain (SEQ ID NO. 4) a VL domainwhich is an amino acid sequence variant of the CAT-212 VL domain, andcombining one or more VL domain amino acid sequence variants thusprovided with one or more VH domains to provide one or more VH/VL domaincombinations; and testing the VH domain amino acid sequence variants orVH/VL combination or combinations for to identify a specific bindingmember that binds eotaxin.
 18. A method of obtaining a specific bindingmember that binds eotaxin, which method comprises: providing startingnucleic acids encoding one or more VH domains which either comprise aCDR3 to be replaced or lack a CDR3 encoding region, and combining saidstarting nucleic acid with a donor nucleic acid encoding the VH CDR3amino acid sequence of SEQ ID NO. 7 such that said donor nucleic acid isinserted into the CDR3 region in the starting nucleic acid, so as toprovide a product nucleic acids encoding VH domains; or providingstarting nucleic acids encoding one or more VL domains which eithercomprise a CDR3 to be replaced or lack a CDR3 encoding region, andcombining said starting nucleic acid with a donor nucleic acid encodingthe VL CDR3 amino acid sequence of SEQ ID NO. 10 such that said donornucleic acid is inserted into the CDR3 region in the starting nucleicacid, so as to provide a product nucleic acids encoding VL domains;expressing the nucleic acids of said product nucleic acids encoding VHdomains and optionally combining the VH domains thus produced with oneor more VL domains to provide VH/VL combinations, and/or expressing thenucleic acids of said product nucleic acids encoding VL domains andcombining the VL domains thus produced with one or more VH domains toprovide VH/VL combinations; selecting a specific binding membercomprising a VH domain or a VH/VL combination that binds eotaxin; andrecovering said specific binding member that binds eotaxin and/ornucleic acid encoding the specific binding member that binds eotaxin.19. A method according to claim 17 or claim 18, further comprisingtesting the specific binding member that binds eotaxin for ability toneutralise eotaxin.
 20. A method according to claim 19 wherein aspecific binding member that binds and neutralises is obtained.
 21. Amethod according to any one of claims 17 to 20 wherein the specificbinding member that binds eotaxin is an antibody fragment comprising aVH domain and a VL domain.
 22. A method according to claim 21 whereinthe antibody fragment is an scFv antibody molecule.
 23. A methodaccording to claim 21 wherein the antibody fragment is an Fab antibodymolecule.
 24. A method according to claim 22 or claim 23 furthercomprising providing the VH domain and/or the VL domain of the antibodyfragment in a whole antibody.
 25. A method according to any one ofclaims 17 to 24 further comprising formulating the specific bindingmember that binds eotaxin or an antibody VH or VL variable domain of thespecific binding member that binds eotaxin into a composition includingat least one additional component.
 26. A method according to any one ofclaims 14 to 24 further comprising binding a specific binding memberthat binds eotaxin to eotaxin or a fragment of eotaxin.
 27. A methodcomprising binding a specific binding member that binds eotaxinaccording to any one of claims 1 to 11 to eotaxin or a fragment ofeotaxin.
 28. A method according to claim 26 or claim 27 wherein saidbinding takes place in vitro.
 29. A method according to any one ofclaims 26 to 28 comprising determining the amount of binding of specificbinding member to eotaxin or a fragment of eotaxin.